EFFECTS OF SODIUM CHLORIDE AND POLYETHYLENE GLYCOL
ON THE WATER RELATIONS, GROWTH, AND MORPHOLOGY
OF CITRUS ROOTSTOCK SEEDLINGS
By
MONGI ZEKRI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987
In the name of God,
Most Gracious,
Most Merciful.
"It is He Who has let free the two bodies
of flowing water: one palatable and sweet
and the other salt and bitter: yet has He
made a barrier between them, a partition
that is forbidden to be passed."
Glorious Quran
Sura XXV (Furquan), or The Criterion
Verse #53
In the name of God
Most Gracious,
Most Merciful.
"It is He Who sendeth down rain from the
skies: with it We produce vegetation of all
kinds: from some We produce green (crops),
out of which we produce grain, heaped up (at
harvest); out of the date-palm and its sheaths
(or spathes) (come) clusters of dates hanging
low and near: and (then there are) gardens of
grapes, and olives, and pomegranates, each
similar (in kind) yet different (in variety):
when they begin to bear fruit, feast your eyes
with the fruit and the ripeness thereof. Behold!
in these things there are signs for people who
believe.
Yet they make the Jinns equals with God, though
God did create the Jinns; and they falsely, having
no knowledge, attribute to Him sons and daughters.
Praise and glory be to Him! (for He is) above
what they attribute to Him!
To him is due the primal origin of the heavens
and the earth: how can He have a son when He hath
no consort? He created all things, and He hath
full knowledge of all things.
That is God, your Lord! There is no god but He,
The Creator of all things: then worship ye Him:
and He hath power to dispose of all affairs."
Glorious Quran
Sura VI (An'am), or Cattle
Verses #99-102
ACKNOWLEDGMENTS
The author expresses his deepest appreciation to his wife, Leila,
for her assistance, encouragement, and patience. He also wishes to
express his sincere gratitude to his mother and to all the family in
Tunisia for their patience and understanding through the years the
author was away from home.
The author expresses his profound gratitude to Dr. L.R. Parsons,
chairman of the supervisory committee, for his valuable advice and
helpful suggestions in the course of conducting the research and in the
preparation of the manuscript.
Sincere thanks re extended to Dr. R. C. J. Koo and to Dr. W. S.
Castle for their advice and for providing greenhouse space.
A special debt of gratitude is acknowledged to Dr. D. L. Myhre and
to Dr. A. G. Smajstrla for their helpful suggestions and comments and
for kindly serving on the supervisory committee.
The author is also grateful to Dr. J. P. Syvertsen and Mr. M. L.
Smith, Jr., for providing equipment and for the use of their laboratory
facilities.
The author's most sincere gratitude is extended to the coordinators
of the Tunisia Agricultural Technology Transfer Project for continuous
encouragement and financial support.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES ix
ABSTRACT xii
INTRODUCTION 1
LITERATURE REVIEW 3
Salts 3
Mechanisms of Salt Tolerance in Plants 3
Mechanisms of Salt Injury 4
Osmotic Effect 4
Ion Toxic Effect 5
Nutritional Imbalance 6
Plant Responses to Salinity 7
Salinity and Citrus 8
Citrus Salinity Research 8
Citrus Tolerance to Salinity 11
Scion 11
Rootstock 11
Salt exclusion 12
Ion concentration 12
Citrus Responses to Saline Conditions 13
Photosynthesis 13
Yield 14
Leaf injury 14
Salinity and high water table 15
Irrigation 15
Reducing Salt Damage 17
Role of Calcium 17
Genetic Improvement 18
MATERIALS AND METHODS 20
General Procedures 20
Experiment 1: Effects of NaCl and PEG on the Root
Conductivity and Leaf Ion Content of Seedlings
of 7 Citrus Rootstocks 21
Experiment 2: Water Relations of Sour Orange and Cleopatra
Mandarin Seedlings under NaCl and PEG Stresses 26
Page
Experiment 3: Fibrous Root Density and Distribution of Sour
Orange Seedlings under NaCl and PEG Stresses 28
Experiment 4: Response of Split-Root Sour Orange Seedlings
to Salinity 29
Experiment 5: Effects of Calcium on Sour Orange Seedlings
Grown under Saline Conditions 32
RESULTS 34
Experiment 1: Effects of NaCl and PEG on the Root
Conductivity and Leaf Ion Content of Seedlings
of 7 Citrus Rootstocks 34
Experiment 2: Water Relations of Sour Orange and Cleopatra
Mandarin Seedlings under NaCl and PEG Stresses 49
Experiment 3: Fibrous Root Density and Distribution of Sour
Orange Seedlings under NaCl and PEG Stresses 57
Experiment 4: Response of Split-Root Sour Orange Seedlings
to Salinity 65
Experiment 5: Effects of Calcium on Sour Orange Seedlings
Grown under Saline Conditions 73
Comparison of Citrus Seedling Responses to NaCl and PEG
Treatments 79
DISCUSSION 82
Leaf Ion Content and Salinity Tolerance 82
Rootstock Tolerance 82
Ion Exclusion and Accumulation 83
Leaf Ion Content and Ion Toxicity 84
Importance of Calcium under Saline Conditions 85
Physiological Effects of NaCl and PEG 86
Effect of NaCl on Root Conductivity 86
Effect of PEG on Root Conductivity 87
Effect of NaCl on Stomatal Conductance 88
Effect of PEG on Stomatal Conductance 89
Effect of NaCl and PEG on Chlorophyll 89
Effect of NaCl on Leaf Thickness and Succulence .... 90
Growth of Citrus Rootstock Seedlings under NaCl and
PEG Stresses 90
Relationship of Leaf Damage Symptoms to
Growth Reduction 91
Root Growth and Distribution under NaCl
and PEG Stresses 91
Effects of Non-Uniform Salinity and Water Stress .... 92
Comparative Effects Between NaCl and PEG 93
SUMMARY AND CONCLUSIONS 95
APPENDIX 100
LITERATURE CITED 116
BIOGRAPHICAL SKETCH 132
LIST OF TABLES
Table
Page
1. Salt treatments and chemical properties of the different
salt treatments 33
2. Shoot dry weight of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 35
3. Root dry weight of seedlings of 7 rootstocks grown
for 5 months under different NaCl and PEG
concentrations 36
4. Specific fibrous root weight of seedlings of 7
rootstocks grown under different NaCl concentrations . . 38
5. Root length, root conductivity, water flow rate, and
osmotic potential of root exudate of seedlings of 7
rootstocks under non-stressed conditions 40
6. Visible injury in seedlings of 7 rootstocks after
5 months of NaCl treatments 42
7. Leaf sodium content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 44
8. Leaf chloride content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 45
9. Ion exclusion and accumulation in leaves of citrus
rootstock seedlings 47
10. Leaf calcium content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 48
11. Monthly new flush growth--area/leaf — of sour
orange seedlings 50
Table Page
12. Monthly new flush growth-leaf number--of sour
orange seedlings 51
13. Leaf succulence of seedlings of 2 rootstocks grown
for 6 months under different NaCl and PEG
concentrations 54
14. Total chlorophyll of seedlings of 2 rootstocks
grown for 6 months under different NaCl and PEG
concentrations 55
15. Fibrous root length in the 3 compartments of the
root boxes for seedlings under different NaCl and PEG
concentrations 63
16. Shoot and root dry weight of split-root sour orange
seedlings under NaCl and PEG stresses 66
17. Midday leaf water, osmotic, and turgor potentials of
split-root sour orange seedlings under NaCl and PEG
stresses 70
18. Midday stomatal conductance and transpiration of
split-root sour orange seedlings under NaCl and PEG
stresses 71
19. Root and shoot dry weight of sour orange seedlings
under different salt treatments 75
20. Total plant dry weight and leaf succulence of sour
orange seedlings under different salt treatments .... 76
21. Leaf mineral analysis of sour orange seedlings under
different salt treatments 77
22. Summary of citrus rootstock responses to NaCl and
PEG as compared to a no salt control 80
23. Shoot root ratio of seedlings of 7 rootstocks grown
for 5 months under different NaCl and PEG
concentrations 100
24. Total plant dry weight of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 101
Table
Page
25. Stem cross sectional area of seedlings of 7
rootstocks grown for 5 months under different NaCl
and PEG concentrations 102
26. Leaf magnesium content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 103
27. Leaf potassium content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 104
28. Leaf phosphorus content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 105
29. Leaf zinc content of seedlings of 7 rootstocks grown
for 5 months under different NaCl and PEG
concentrations 106
30. Leaf manganese content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations 107
31. Seedling height of seedlings of 2 rootstocks grown
for 6 months under different NaCl and PEG
concentrations 108
32. Total leaf area of seedlings of 2 rootstocks grown
for 6 months under different NaCl and PEG
concentrations 109
33. Specific leaf weight of seedlings of 2 rootstocks
grown for 6 months under different NaCl and PEG
concentrations 110
LIST OF FIGURES
Figure
1. Osmotic potential versus NaCl concentration as
determined by vapor pressure (VPD) and freezing
point depression (FPD) 22
2. Osmotic potential versus PEG concentration as
determined by vapor pressure (VPD) and freezing
point depression (FPD) 23
3. Sour orange seedlings with a split-root system 30
A. Effect of 3 NaCl concentrations on the total
fibrous root length, root hydraulic conductivity,
and water flow rate for seedlings of 7 citrus
rootstocks 37
5. Relationship between root hydraulic conductivity
and specific root weight of seedlings of 7 citrus
rootstocks under non-stressed conditions Al
6. Effect of NaCl at an osmotic potential of -0.35
MPa on the 7 rootstocks after 5 months of
salinity treatments A3
7. Relationship between water flow rate and osmotic
potential of root exudate of sour orange and
Cleopatra mandarin seedlings 53
8. Relationship between midday stomatal conductance
and root conductivity of sour orange and Cleopatra
mandarin seedlings 56
9. Midday stomatal conductance of sour orange
seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCl or PEG ... 58
10. Relationship of time of day to stomatal
conductance of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or
with added NaCl or PEG during 2 consecutive days .... 59
Figure
11. Growth of sour orange seedlings irrigated with
nutrient solution containing no salt (NS) or with
added NaCl or PEG 60
12. Fibrous root length of sour orange seedlings
irrigated with nutrient solution containing no
salt (NS) or with added NaCl or PEG 61
13. Fluctuations in shoot and root growth of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCl or PEG ... 62
14. Root density and distribution of sour orange
seedlings growing in root boxes under
non-stressed (NS) and stressed (NaCl, PEG)
conditions 64
15. Root development of split-root sour ornage
seedlings under uniform and non-uniform NaCl and
PEG stress 67
16. Leaf water, osmotic, and turgor potential of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with NaCl added to both
root halves 69
17. Relationship between transpiration and stomatal
conductance of sour orange seedlings 72
18. Cross sections of sour orange leaves 74
19. Sour orange leaves from non-stressed (control)
and stressed (NaCl, PEG) seedlings 81
20. Effect of 3 NaCl concentrations on the osmotic
potential of root exudate collected from
seedlings of 7 citrus rootstocks Ill
21. Relationship of time of day to stomatal
conductance of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or
with added NaCl during 3 consecutive days 112
22. Relationship of time of day to stomatal
conductance of Cleopatra mandarin seedlings
irrigated with nutrient solution containing no
salt (NS) or with added NaCl during 3 consecutive
days 113
Figure Page
23. Relationship of time of day to stomatal
conductance of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or
with added PEG during 3 consecutive days 114
24. Relationship of time of day to stomatal
conductance of Cleopatra mandarin seedlings
irrigated with nutrient solution containing no
salt (NS) or with added PEG during 3 consecutive
days H5
Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EFFECTS OF SODIUM CHLORIDE AND POLYETHYLENE GLYCOL
ON THE WATER RELATIONS, GROWTH, AND MORPHOLOGY
OF CITRUS ROOTSTOCK SEEDLINGS
By
MONGI ZEKRI
December 1987
Chairman: Dr. Lawrence R. Parsons
Major Department: Horticultural Science (Fruit Crops)
The effects of sodium chloride (NaCl) and polyethylene glycol (PEG)
on the growth, water relations, and leaf mineral content of citrus
rootstocks were investigated. Significant growth reduction and
physiological disturbances occurred even at NaCl and PEG concentrations
of -0.10 MPa. Growth reduction and physiological changes were found to
precede visible damage. Growth was reduced up to 30% without being
accompanied by visible leaf injury symptoms. Leaf burn symptoms
developed only after a threshold value of chloride accumulation (1%) was
reached. Leaf burn symptoms developed too slowly to accurately evaluate
salt damage. Root conductivity correlated better with salinity
tolerance among rootstocks than did total fibrous root length.
Sodium chloride usually caused less damage than PEG. Unlike PEG,
NaCl significantly increased leaf thickness and succulence along with
leaf sodium and chloride concentrations, but reduced calcium and zinc
contents in the leaves. Both NaCl and PEG reduced leaf magnesium and
potassium contents but increased leaf phosphorus and manganese contents.
Differences in sodium and chloride exclusion capacities among
rootstocks were found. Sour orange, rough lemon, and Milam were sodium
and chloride accumulators. Poncirus trifoliata, Swingle citrumelo, and
Carrizo citrange were sodium excluders but chloride accumulators.
Cleopatra mandarin was a chloride excluder but a sodium accumulator.
Differences in NaCl sensitivity among rootstocks were also found.
Cleopatra mandarin and sour orange were the least sensitive, Milam and
Poncirus trifoliata were the most sensitive, and rough lemon, Swingle
citrumelo, and Carrizo citrange were intermediate in sensitivity.
Cleopatra mandarin tolerated high concentrations of NaCl by partial
exclusion of chloride while sour orange tolerated NaCl even though it
accumulated sodium and chloride in its leaves. Sour orange might have
the ability to compartmentalize these ions and exclude them from the
cytoplasm where they may inhibit metabolic processes.
Seedlings receiving NaCl or PEG produced small and shallow root
systems with the majority of the roots occurring in the top layer.
Addition of calcium sulfate to saline irrigation water was found to be
beneficial in overcoming the detrimental effects of NaCl on citrus. The
split-root experiment showed that citrus could withstand substantial
amounts of stress as long as half of the root system was growing in a
non-stressed environment.
INTRODUCTION
It is well established that salt can impair the performance of many
agricultural plants. Salts present in the soil and irrigation water are
a serious problem for commercial agriculture particularly in arid and
semi-arid regions. However, the potential for salinity damage also
exists in humid climates. Controlling or reducing salt injury is
usually achieved either through soil management practices and irrigation
with good quality water or by combining these practices with the use of
salt-tolerant plants.
Citrus is a fruit crop of international significance. It is grown
in over 50 countries and ranks among the top 3 tree fruit crops in world
production. In certain areas where citrus is grown, salinity is already
a problem of some importance. In other areas, the future of
citriculture is threatened by salinity largely because agriculture is
being forced to use lower quality land and water for irrigation. In
agricultural areas with salinity problems, citrus is particularly
vulnerable because there is relatively little salt tolerance in the
genus.
Salinity studies have shown that among species, cultivars, and
various selections, only 2 roots tocks, Cleopatra mandarin and Rangpur
lime, have a limited capacity to tolerate certain salts. However,
rootstocks are usually selected for other attributes such as yield and
fruit quality. Rootstocks deficient in these characteristics are not
likely to be commercially used despite favorable salt tolerance.
1
2
In Florida, there are many citrus plantings located in coastal areas
where saline water is being used for irrigation. Citrus planting in
these and other southern Florida areas has been accelerated by extensive
freeze damage in more northern areas. These changes in the citrus
industry, as well as the diseases triteza and blight, have greatly
affected interest in rootstock characteristics including salt tolerance.
In the past, virtually all evaluations of citrus response to
salinity were based on visual leaf injury and correlations with leaf
chloride content. There were no root system observations recorded and
no detailed physiological studies conducted. Such observations and
measurements of physiological responses are necessary for a complete
understanding of salt injury and tolerance in plants. This information
is particularly valuable for efficient breeding and screening of new
germplasm for salt tolerance.
The objectives of this research are the following:
1. To compare the salt tolerance of citrus rootstocks commercially
important in Florida and to determine which rootstocks are salt
excluders or salt accumulators.
2. To determine the salt concentrations at which growth is
depressed, water balance is disturbed, and leaves are injured.
3. To separate specific ion effects of salts from their osmotic
effects by comparing growth, water relations, and plant chemical
analyses under NaCl and PEG stresses.
4. To measure the effects of several NaCl and PEG concentrations on
root growth and distribution.
5. To study citrus growth and water relations under non-uniform
salinity (split-root system).
6. To examine the importance of calcium in reducing NaCl damage.
LITERATURE REVIEW
Salts
Many hectares of land throughout the world are too saline for
profitable agriculture (Carter, 1975). Large amounts of arable lands
are being removed from crop production every year due to increasing soil
salinity (Chapman, 1975; Epstein et al., 1980). Saline irrigation water
combined with fertilizer application are the factors most responsible
for increasing soil salinity (Epstein et al., 1980; Jones et al., 1952;
Stewart et al., 1977).
The ions in soil waters which contribute significantly to salinity
problems are principalis sodium, chloride, calcium, magnesium, sulfate,
potassium, bicarbonate, carbonate, nitrate, and occasionally borate ions
(Bernstein and Hayward, 1958; Peck, 1975; Shainberg, 1975). However,
most salinity research has involved NaCl because it is the most common
salt in saline soils and irrigation waters.
Mechanisms of Salt Tolerance in Plants
Salt-tolerant plants are generally thought to be protected from salt
stress by either ion accumulation or ion exclusion. Accumulation of
high concentrations of ions in halophyte leaves has been known to be a
salt tolerance mechanism (Flowers et al., 1977; Greenway and Munns,
1980). Salts can be tolerated because ions are compartmentalized in the
vacuole and not in the cytoplasm. Hence, metabolic processes are not
inhibited. These ions in the vacuoles balanced with neutral organic
solutes in the cytoplasm lower the leaf osmotic potential. This allows
3
4
the plant to extract water from saline solutions. However, salt
tolerance in glycophytes (nonhalophy tes) is related to ion exclusion
because of the plant's inability to compartmentalize toxic ions in a
useful way and to adjust osmotically (Greenway and Munns, 1980).
Mechanisms of Salt Injury
Salt damage to plants is caused by the decrease in the water
potential of the soil solution or by the toxicity of specific ions.
Some workers attribute most of the salt damage to osmotic stress
(Bernstein, 1961, 1963; Bernstein and Hayward, 1958; Bielorai et al.,
1978, 1983; Bohn et al., 1979). Others favor the idea that toxic
effects of specific ions predominate in restricting growth and yields
(Babaeva et al., 1968; Gollek, 1973; Strogonov, 1964).
A common method of distinguishing between osmotic and ion toxic
effects of salts is to compare the effects of isosmotic solutions of the
salt with those of non-toxic organic substances. If the salt injury is
simply osmotic, all solutes should produce the same injury at the same
osmotic potential (Levitt, 1980). Polyethylene glycol (PEG), a
non-ionic compound, has been successfully used as an osmoticum for
subjecting plants and plant tissues to known levels of water stress
(Janes, 1966; Kaufmann and Eckard, 1971; Kawasaki et al., 1983a, b).
Osmotic Effect
Water is osmotically more difficult to extract from saline
solutions. Pair et al. (1975) pointed out that the addition of 0.4%
salts had the effect of reducing the total available water in the soil
by approximately 33%. Salt addition is analogous to soil drying since
both result in reduced water uptake. In extreme circumstances, salinity
can prevent water uptake even when the soil is at field capacity (Hartz,
1984). Water uptake by mature grapefruit trees, mature Valencia orange
5
trees, and Valencia orange seedlings was reduced as salinity increased
(Bielorai et al., 1983; Hayward and Blair, 1942; Plessis, 1985).
Ion Toxic Effect
Ion toxic effect of salt is attributed to excess accumulation of
certain ions in plant tissues and to nutritional imbalances caused by
such ions. Ion excess has been defined as a condition where high
internal ion concentrations reduced growth (Greenway and Munns, 1980).
In many crops, salt injury increases with increased salt uptake.
Raspberries were found to accumulate chloride ions more rapidly and
consequently were more severely injured than blackberry (Ehlig, 1964).
Tagawa and Ishizaka (1963) found that the primary cause of injury to
rice by NaCl was chloride accumulation in the shoots. When treated with
NaCl, a less resistant barley variety accumulated higher levels of
chloride and sodium than a more resistant variety (Greenway, 1962).
Salt damage to citrus has been mainly attributed to excessive
accumulation of chloride and sodium in the leaves (Abdel-Messih et al.,
1979; Chapman et al., 1969; Cooper, 1961; Cooper et al., 1951; 1952b;
Cooper and Peynado, 1953; El-Azab et al., 1973; Furr and Ream, 1968;
Grieve and Walker, 1983). Goell (1969) suggested that salt ions such as
chloride in citrus leaves might shorten the life span of leaves by
increasing chlorosis (loss of chlorophyll and photosynthetic potential)
and by promoting senescence and abscission. Sulfate and other ions also
caused damage to citrus (Bhambota and Kanwar, 1970; Bingham et al.,
1973; Cerda et al. 1979; Hewitt and Furr, 1965a; Peynado and Young,
1964). It has been suggested that the accumulation of ions in large
amounts in the leaves is the main factor causing leaf burn and
inhibition of certain metabolic processes.
6
Sodium can also cause injury to plants through its deleterious
effect on the soil. When the proportion of exchangeable sodium is
relatively high, clay particles in the soil tend to disperse and -block
the pores through which water flows. This phenomenon decreases the
hydraulic conductivity of the soil (Bohn et al., 1979; Shainberg, 1975)
and causes poor aeration. Studies by Aldrich et al. (1945) demonstrated
that inferior performance of orange trees was caused primarily by poor
water penetration resulting from sodium accumulation on the exchange
complex.
Nutritional Imbalance
Salt can also damage plants by causing nutritional imbalances. High
sodium levels can lead to calcium and magnesium deficiencies (Bohn et
al., 1979). In spinach and lettuce, sodium salts decreased dry matter
production as well as leaf potassium, magnesium, and calcium contents
(Matar et al., 1975). Pumpkin and sweet clover plants subjected to NaCl
showed potassium deficiency (Solov'ev, 1969). A decrease in potassium
uptake at higher concentrations of sodium was found in sugarcane
(Nimbalker and Joshi, 1975) and rice (Paricha et al., 1975). With
increased salinity, potassium and phosphorus uptake decreased in grapes,
guava, and olive plants (Taha et al., 1972), in wheat (Sharma and Lai,
1975), and in barley (Kawasaki et al., 1983b).
In citrus, nutritional imbalance has been also attributed to
depressed absorption of some nutrients. A decrease in the concentration
of calcium, magnesium, and sometimes potassium was found when salt
concentration in the irrigation water was increased (Jones et al., 1957;
Patil and Bhambota, 1980; Pearson et al., 1957).
7
Plant Responses to Salinity
Salinity has been known to adversely affect all stages of plant
development such as germination, vegetative growth, and fruiting.
Salinity has also been found to depress chlorophyll content,
photosynthesis, stomatal conductance, root conductivity, and
transpiration of many crops. For example, growth of citrus (Furr and
Ream, 1968), Vicia faba (Helal and Mengel, 1981), pepper (Hoffman et
al., 1980), alfalfa (Keck et al., 1984), bean (Meiri and
Poljakoff-Mayber, 1970), and corn (Siegal et al., 1980) was
significantly depressed under saline conditions.
Yield of grapefruit (Bielorai and Levy, 1971; Bielorai et al., 1978,
1983), orange (Bingham et al., 1973, 1974; Chapman et al., 1969), celery
(Francois and West, 1982), and muskmelon (Shannon and Francois , 1978)
was severely reduced due to salinity stress. Salinity was found to
alter fruit quality by decreasing the "pack out" of oranges at a
commercial packing shed (Bingham et al., 1974) and by decreasing the
marketable yield of tomato and melon (Mizrahi and Pasternak, 1985;
Shannon and Francois, 1978). It was found that the relative amount of
the premium grade fruit decreased with use of saline water even though
there was a trend toward higher soluble solids and better taste (Bingham
et al., 1974; Mizrahi and Pasternak, 1985; Shannon and Francois, 1978).
Salinity reduced leaf chlorophyll content in grapevine, bean,
barley, citrus and mangrove (Downton and Millhouse, 1985), spinach
(Downton et al., 1985), and Acacia saligna (Shaybany and Kashirad,
1978). Leaf chlorophyll content declined only when certain amounts of
salt ions accumulated in the leaves. Salinity reduced photosynthesis in
spinach (Downton et al., 1985), rice (Flowers et al., 1985), Xanthium
strumarium (Schwarz and Gale, 1983), beans (Seemann and Critchley,
8
1985), and Acacia saligna (Shaybany and Kashirad, 1978). Under most
circumstances, photosynthetic reduction was attributed to ion
accumulation in the leaves and to reduction in stomatal conductance.
Salinity was found to reduce root conductivity in white lupin (Munns
and Passioura, 1984) and beans (O'Leary, 1969; 1974). However, salinity
did not affect root conductivity in barley (Munns and Passioura, 1984),
sunflower and tomato plants (Shalhevet et al., 1976). Reduced hydraulic
conductivity of roots has been attributed to root suberization and to
reduced root membrane permeability.
Salinity and Citrus
Citrus is generally classified as a salt sensitive crop because
physiological disturbances and growth and fruit yield reductions can
occur at relatively low salinity levels (Bernstein, 1969; Bielorai et
al., 1978, 1983; Boaz, 1978; Cherif et al., 1982; Cooper and Shull,
1953; Francois and Clark, 1980; Furr e< al., 1963; Kirkpatrick and
Bitters, 1969; Marsh, 1973; Patil and Bhambota, 1980; Pehrson et al.,
1985; Walker et al., 1982).
Citrus Salinity Research
The response of citrus to salinity is a topic of concern in many
regions where citrus is grown especially the United States, Israel,
Egypt, India, Spain, and Tunisia. In the United States, salinity
studies essentially began in Texas during the 1940s. Investigations
were led by U.C. Cooper with emphasis on differences in salinity
tolerance among citrus rootstocks (Cooper, 1948; Cooper and Gorton,
1952; Cooper and Peynado, 1959; Cooper and Shull, 1953; Cooper et al.,
1951). The work was conducted mostly on young budded trees grown in the
field. Salinity treatments consisted of NaCl + CaCl added to Rio
Grande river water. These studies led to the observation that chloride
9
exclusion was strongly correlated with salt tolerance. Chloride
accumulation or exclusion and leaf injury symptoms were used to classify
salt tolerant and salt sensitive rootstocks.
Salinity studies on citrus were started in California in the 1950's
(Harding et al., 1958a; Janes et al., 1952; Pearson and Goss, 1953), in
Israel in the 1970s (Bielorai et al., 1973; Heller et al., 1973), and in
Australia in the 1970s (Cole and Till, 1977). Most of these studies
were conducted in the field on mature citrus trees and were focused on
yield reduction and fruit quality alteration as a function of salt
concentration in irrigation waters (Bielorai et al., 1978, 1983; Bingham
et al., 1973, 1974; Boaz, 1978; Francois and Clark, 1980; Levy et al.,
1979; Pehrson et al., 1985; Shalhevet et al., 1974).
Recent salinity work in Israel was directed to plant breeding using
cell culture techniques (Ben-Hayyim and Kochba, 1983; Ben-Hayyim et al.,
1985). Recent work in Australia was conducted mainly with young
rootstock seedlings grown in pots under glasshouse conditions
(Behboudian et al., 1986; Grieve and Walker, 1983: Walker and Douglas,
1983; Walker et al., 1982, 1983, 1984, 1986). Salinity treatments
consisted of NaCl added to a nutrient solution. These studies were
focused on sodium and chloride exclusions mechanisms, water relations,
and photosynthesis. Photosynthesis was severely reduced and
photosynthetic reduction was attributed to a loss of turgor in salt
excluder rootstocks and to chloride accumulation in salt accumulator
rootstocks.
Some salinity work on citrus conducted in Egypt (Abdel-Messih et
al., 1979; Minessy et al., 1973), India (Bhambota and Kanwar, 1970;
Patil and Bhambota, 1980), Spain (Cerda et al., 1979; Guillen et al.,
1978), and Tunisia (Cherif et al., 1981; 1982; Zid, 1975; Zid and
10
Grignon, 1985, 1986) on budded trees and rootstock seedlings involved
ion analysis and nutrient absorption. These studies showed that
salinity caused nutritional imbalance, growth reduction, and leaf- burn.
Growth reduction was attributed to potassium deficiency and foliar
necrosis to sodium accumulation in the leaves.
Salinity is of increasing concern in Florida. Salt water intrusion
into groundwater in areas where citrus is grown has increased the need
for salinity studies in Florida. Many citrus rootstocks are being used
in Florida such as sour orange, Swingle citrumelo, Carrizo citrange, and
Milam without knowing their salt tolerance. As a result, there is an
incentive to study the salinity tolerance of these and other rootstocks
which are commercially important.
Physiologists often concentrate on the activities of shoots and
neglect roots because they are out of sight and more difficult to study
than shoots (Kramer, 1983). Roots play an important role in the growth
and development of the entire plant. Their health, vigor and activity
can be an index of the functioning of the above-ground parts (Crider,
1927). It is important to investigate root growth and distribution
because roots are directly in contact with salts in the soil. Detailed
information on the growth behavior and morphological development of
citrus root systems under salt conditions is not available.
The two major resistances to water movement through the
soil-plant-atmosphere continuum are the roots and the stomata (Kramer,
1969; Kriedemann and Barrs, 1981). Root conductivity and stomatal
conductance are important variables to be monitored in salinity studies
because they can provide information on the water balance disturbance
caused by salt. Root conductivity of some in citrus rootstocks under
salinity stress has not been previously studied. Furthermore,
11
information relating root conductivity to stomatal conductance and
transpiration as a function of different osmotic concentrations is
lacking.
Under field conditions, the roots of an individual plant grow in
soil which varies in water content and salt concentration both in space
and with time. In assessing the suitability of water for irrigation, it
is usually assumed that plants respond to the mean root zone salinity
(Shalhevet and Bernstein, 1968). However, some workers suggest that the
least saline part of the rooting zone controls the overall plant growth
and yield (Lunin and Gallatin, 1965). Responses of citrus to
non-uniform salinity or to zonal salinization are not known.
Citrus Tolerance to Salinity
Scion. Differences in salt tolerance among citrus varieties or
scions have been shown. Boaz (1978) concluded that Valencia orange had
a lower tolerance to salinity than grapefruit on sweet orange rootstock.
Bernstein (1969) reported that lemon was more sensitive to salinity than
orange which was more sensitive than grapefruit. Miwa et al. (1957)
also found that lemon was the most susceptible variety to foliar spray
injury from sea water. Results of Pearson and Huberty (1959) showed
that navel orange trees were more sensitive to irrigation water quality
than Valencia orange trees. Budded on rough lemon, salt tolerance
decreased in the following order: Hamlin, Valencia, Pineapple and Blood
red sweet orange (Bhambota and Kanwar, 1969). Valencia seemed to be
more sensitive to salinity than Shamouti when both were grafted on sour
orange rootstock (Shalhevet et al., 1974).
Rootstock. Some studies have indicated a wide range in salt
tolerance among citrus rootstocks (Cooper, 1948; Cooper and Edwards;
1950; Coopei et al., 1952b, 1958). Cooper et al. (1951) found that
12
Cleopatra mandarin and Rangpur lime are relatively salt-tolerant
rootstocks. They classified sour orange, rough lemon, sweet lemon,
tangelo and sweet lime as sensitive rootstocks and Florida sweet .orange
and trifoliate orange as very sensitive. In another study, Cleopatra
mandarin and Rangpur lime were also found to be the most tolerant
rootstocks and Carrizo citrange was the most sensitive rootstock (Joolka
and Singh, 1979; Patil and Bhambota, 1978). Trifoliate orange and rough
lemon were found to be very salt sensitive (Bhambota and Kanwar, 1969).
Although some selections of sour orange differed in salt tolerance, Ream
and Furr (1976) found that none of them was as salt tolerant as
Cleopatra mandarin.
Salt Exclusion. Exclusion of certain ions has been demonstrated in
some citrus rootstocks. Rangpur lime and Cleopatra mandarin appear to
be chloride excluders (Cooper, 1961; Cooper and Gorton, 1952; Cooper and
Peynado, 1959; Douglas and Walker, 1983; Grieve and Walker, 1983; Hewitt
and Furr, 1965b; Walker, 1986; Walker et al., 1983; Wutscher et al.,
1973). Trifoliate orange appears to be a sodium excluder (Elgazzar
et al., 1965; Grieve and Walker, 1983; Walker, 1986) and Citrus
macrophylla a boron excluder (Cooper and Peynado, 1959; Embleton et al.
1962). This suggests the existence of a blocking mechanism in the
transport of these ions (Fernandez et al., 1977). It also indicates the
existence of apparently separate mechanisms which regulate the uptake
and transport of ions (chloride and sodium) in salt-stressed citrus
(Fernandez et al., 1977; Grieve and Walker, 1983; Walker et al., 1983).
Ion concentration. Citrus is a nonhalophyte, and its tolerance to
salinity is correlated with its ability to restrict the entry of ions
into the shoots (Greenway and Munns, 1980). Injury to citrus from NaCl
has been attributed to excess chloride accumulation (Ben-Hayyim and
13
Kochba, 1983; Cooper, 1961; Cooper and Gorton, 1952; Furr and Ream,
1969). In an effort to screen young citrus trees for salt tolerance,
Hewitt et al. (1964) found that the leaves could be analyzed for
chloride after 3 to 4 weeks of treatment with highly saline irrigation
water. Fernandez et al. (1977) considered foliar chloride content as a
suitable index of the soil salinity status and toxicity levels.
However, Ben-Hayyim et al. (1985) showed the difficulty in determining
if any particular ion could serve as a reliable marker for salt
tolerance in citrus.
Citrus Responses to Saline Conditions
Photosynthesis. Photosynthetic rates were reduced by 50 to 75%
after 70 days of NaCl stress (Behboudian et al., 1986; Walker et al.,
1982). A decrease in photosynthesis is often caused by a drop in leaf
turgor, but studies have shown different turgor responses to salinity.
In one study with Rangpur lime, photosynthesis reduction was attributed
to low turgor pressures in rangpur lime and not to leaf chloride or
sodium concentrations since there was no significant difference in
concentrations of these ions between salt-stressed and control leaves.
In contrast to Rangpur lime, photosynthetic reduction during salt
treatment in Etrog citron was associated with a marked increase in leaf
chloride since turgor was not reduced. Their work established that a
plant's capacity for salt exclusion alone or turgor maintenance alone
was unable to protect citrus seedlings against photosynthetic reduction.
Therefore, to improve salt tolerance in citrus, studies need to be
focused not only on salt exclusing rootstocks but also on the ability of
scions to maintain turgor. It appears that the inability to osmotically
adjust and exclude toxic ions is related to citrus sensitivity to
salinity (Zid and Grignon, 1986).
14
Yield. Citrus yield has been related to salt concentration in the
soil (Bielorai et al., 1978; Harding et al., 1958b). According to Boaz
(1978) and Maas and Hoffman (1977), the threshold salinity is an •
electrical conductivity of the soil saturation extract of 1.8 dS/m (1.8
mmhos/cm) for oranges and grapefruit. Above this threshold, yield is
reduced at a rate of 16% per dS/m. Pehrson et al. (1985) stated that 10
and 50% yield reductions for citrus were associated with electrical
conductivities of the soil saturation extract of 2.3 and 4.8 dS/m,
respectively.
Salinity was found to significantly reduce citrus yield without
visual symptoms (Pehrson et al., 1985). The use of moderately saline
irrigation water (2.5 dS/m) decreased orange yield by about 30% without
any visible leaf injury symptoms (Bingham et al., 1974). Within a
concentration range of 2 to 2.7 dS/m, 9 to 18% yield reduction in
grapefruit occurred without apparent tonicity symptoms (Bielorai et al.,
1978, 1983). When irrigated with moderately saline water (15 to 30 mM,
CaCl + Na,S0 + MgS04 ) , Valencia orange had yield reductions of 34 to
54% with no visible leaf injury symptoms (Francois and Clark, 1980).
Leaf injury. Salinity effects develop slowly so that leaf injury
symptoms appear after a certain period of time. However, the length of
this time period is shortened by higher salt concentrations. Grown in
the field, two-year-old Ruby red grapefruit on sour orange rootstock
irrigated with salt solutions of 2500 mg/L (50:50 NaCl and CaCl2) showed
no visible symptoms of salt injury during a one year period. Trees
irrigated with 4000 mg/L salt solution developed leaf bronzing within
1 month and marginal burning of the leaves within 2 months. Trees
irrigated with 5000 mg/L salt solution were completely defoliated within
a one year period (Cooper, 1961; Cooper et al., 1952a).
15
Salinity and high water table. Relatively few studies have been
conducted to investigate the effects of a combination of water table and
salinity on citrus even though this condition exists in many part-s of
the world. Studying the effects of salinity and water table on the
growth and mineral composition of young grapefruit trees, Pearson and
Goss (1953) found that the rates of defoliation and twig dieback due to
salinity were greatly accelerated by a frequently fluctuating water
table. In a more detailed report of the same study, Pearson et al.
(1957) concluded that the salinity factor accounted for approximately
90% of the variance in growth while the water table factor accounted for
only about AX. They found that sodium and chloride accumulated in toxic
amounts in the leaves and were responsible for the decrease in growth.
However, while investigating the effect of different water table depths
and salinity levels on sweet orange, Kanwar and Bhambota (1969) observed
that the adverse effect of water table was more pronounced than that of
salinity. Both studies agreed that the interaction of water table and
salinity affected the trees more severely than either condition alone.
The fact that Cleopatra mandarin is more sensitive to flooding
(Ford, 1964) but more salt tolerant (Cooper et al., 1951) than sour
orange raises the question about the performance of these two rootstocks
under saline conditions associated with high water table or flooding
problems.
Irrigation. Citrus is relatively sensitive to salinity, but can
withstand high salt concentrations depending on the variety, rootstock,
and irrigation management. Good irrigation management should consider
the salinity factor in the irrigation water, in the soil, and in the
root zone (Boaz, 1978). Methods of irrigation scheduling which do not
account for salinity are not sufficiently accurate for scheduling
16
irrigation in areas with a saline high water table. Irrigation water
containing about 250 mg chloride per liter reduced grapefruit yield by
28 to 322 when trees were irrigated at intervals of 40 days compared to
intervals of 18 days (Bielorai and Levy, 1971; Bielorai et al., 1973).
These studies demonstrated that the effect of salinity is more severe at
lower soil water content.
Overhead sprinkler irrigation should be avoided when using water
containing high levels of salts because salt residues can accumulate on
the foliage and seriously injure plants. Navel orange accumulated
injurious amounts of chloride and sodium from sprinkler-applied water
having 500 to 900 ppm total dissolved solids (Harding et al., 1958a).
Considerable leaf burn and defoliation of these trees were found to be
correlated with excessive amounts of sodium and chloride and lower
amounts of potassium in the leaves. Leaf injury of navel orange trees
developed at concentrations of 5 to 10 mmol/L of NaCl, CaCl2 or Na2S04
in the sprinkler-applied waters (Ehlig and Bernstein, 1959). Salt
content of up to 1300 mg/L caused defoliation of sprinkler-irrigated
citrus trees in Texas (Lyons, 1977). In Australia, during periods of
high salinity in the irrigation water, foliar absorption of sodium and
chloride occurred when using overhead sprinklers on citrus. It was
believed that this problem caused poor tree health, low yield, and
possibly poor fruit quality in citrus (Cole and Till, 1977).
Frequency rather than duration of sprinkler irrigation is perhaps
more important in foliar absorption of salts. Salt injury was higher
under higher evaporation conditions and with short and frequent periods
of overhead sprinkling (Eaton and Harding, 1959; Ehlig and Bernstein,
1959; Harding et al., 1958a).
17
Micro-irrigation is gaining in popularity not only in arid regions
but also in humid subtropical areas. Micro-irrigation refers to both
drip and microsprinkler irrigation. Micro-irrigation enables the use of
poorer quality water that cannot be tolerated with overhead sprinklers.
Direct foliar uptake of salts, and hence leaf injury, is avoided with
drip irrigation (Calvert and Reitz, 1966). Nevertheless, saline water
cannot be used indiscriminately with micro-irrigation systems.
Comparative studies between overhead sprinklers and drip systems using
saline water showed that vegetative growth, root development, and yield
were greater with drip than with sprinkler irrigation (Goldberg and
Shmueli, 1971; Shmueli and Goldberg, 1971). In a comparison of flood
and drip systems, water high in chloride and boron was applied to young
grapefruit trees on many rootstocks (Vutscher et al., 1973). More
chloride and boron accumulation was found in trees that were flood
irrigated than in thoc that were micro-irrigated.
Drip irrigation at frequent intervals maintains a low soil water
tension and prevents salt accumulation within the wetted zone.
Consequently, water with higher salinity levels may be used without
significantly affecting the yield. Nevertheless, salt accumulation
under drip irrigation must be considered because salts may accumulate
both at the periphery of the wetted zone and on the soil surface
(Bielorai, 1977, 1985; Goldberg et al., 1976; Hoffman et al., 1985;
Yaron et al., 1973).
Reducing Salt Damage
Role of Calcium
Calcium has been known to have an ameliorating effect on the growth
of plants under saline conditions (Deo and Kanwar, 1969; Epstein, 1972;
Hyder and Greenway, 1965). This effect has been attributed to calcium
18
preventing the uptake of the sodium ion to injurious levels, and
allowing the uptake of potassium (Uaisel, 1962). In the presence of
adequate concentrations of calcium, bean plants were able to exclude
sodium and to withstand the effects of relatively high NaCl
concentrations (LaHaye and Epstein, 1969, 1971). In barley, inhibition
of the absorption and translocation of potassium and phosphorus by NaCl
was found to recover dramatically in the presence of calcium (Kawasaki
et al., 1983b). Application of gypsum to the soil or in the irrigation
water markedly reduced the percentage of soluble sodium in the soil
(Harding et at., 1958b) and reduced the percentage of sodium in citrus
leaves and roots (Jones et al., 1952; Pearson and Huberty, 1959).
Calcium amendments are commonly used for replacement of exchangeable
sodium (Richards, 1954). Calcium can flocculate soil in which clay
particles and aggregates have been dispersed by sodium. Salt-affected
soils can therefore be made productive by chemical amendment, drainage,
and irrigation with high quality water, but sometimes the cost of these
operations exceeds the expected returns from the land.
Genetic Improvement
In recent years, adapting plants to saline environments through
breeding and genetic manipulation have been attempted (Epstein et al.,
1980). The genetic basis for salt tolerance, using information from
studies with whole plants, has allowed the identification of plants with
increased salt tolerance. Another approach is to increase salt
tolerance through cell culture (Croughan et al., 1981).
In some species, the variability in salt tolerance may not be
adequate for a successful breeding program because it may not be
possible to find salt- tolerant wild relatives and use them as sources of
germplasm. Suspension of cells from salt-sensitive plants in solutions
19
having various degrees of osmotic stress was found to be a promising
technique to select salt-tolerant cells from salt-sensitive cells. This
implies that the genetic information for growth in a saline environment
may be present in salt-sensitive cells but is not expressed. Selection
of salt-tolerant cells may provide genetic material that will help
improve our understanding of salinity resistance at the cellular level.
MATERIALS AND METHODS
General Procedures
This study consisted of 5 experiments involving citrus seedlings
grown in greenhouses in central Florida. Seeds were sown in plastic
trays composed of individual cells. The trays were filled with PROMIX
BX [60% Canadian peat, 20% perlite, and 20% vermiculite with dolomitic
limestone, superphosphate, calcium nitrate and fritted trace elements
added]. The seeds were irrigated with tap water twice a week until
emergence. Seedlings were irrigated with tap water every other day and
fertilized with 20-20-20 (N,P,K) fertilizer once a week. The
temperature and relative humidity in the greenhouses were controlled by
both heating and evaporative cooling systems with conventional end-wall
air circulation fans. The minimum and maximum temperature and relative
humidity ranged from 20 to 35°C and from 40 to 100%, respectively.
Three to 6 months after emergence, uniform seedlings were selected
and transplanted into pots or wooden boxes containing fine sand taken
from the top 30 cm of a citrus orchard soil. The soil was Astatula fine
sand (hyperthermic, uncoated Typic Quartzipsamments) with a pH of 6.5
and a field capacity and a wilting percentage of 7.2% and 1.2% (volume
basis), respectively. Seedlings were irrigated every 2 to 3 days with
half strength Hoagland's solution #1 (Hoagland and Arnon, 1950) for at
least one month before starting salt and polyethylene glycol (PEG)
treatments. Treatments were started by adding NaCl, PEG, or other salts
to the Hoagland solution.
20
21
The water holding capacity of the soil in the containers was about
18£ (volume basis). The irrigation frequency was 2 to 3 days. The
amount of solution added each time was based on bringing the soil to
slightly more than the water holding capacity of the soil in the
containers to prevent salt accumulation in the growth medium and to
prevent plants from undergoing a drought stress.
Standard curves (Fig. 1, 2) of osmotic potential versus solute
concentration were developed for NaCl and PEG 4000 by measuring vapor
pressure and freezing point depression. The values obtained were
similar to those of Steuter et al. (1981) who compared freezing point
depression and vapor pressure methods for determination of water
potential of PEG solutions. Electrical conductivities of the different
treatments were determined with a conductivity meter. Electrical
conductivity values were converted to TDS (Richards, 1954).
Sodium chloride and PEG treatments were continued for at least 4
months, after which the plants were harvested and the roots were washed
briefly with tap water to free them of sand particles. Shoots were
separated into stems and leaves, and roots were separated into taproots,
lateral roots, and fibrous roots (roots less than 2 mm in diameter).
The material was oven-dried for 3 days at 60°C, weighed, ground, and
retained for ion analysis.
Analysis of variance (F-test) was used to determine significant
differences and Duncan's multiple range test was employed for mean
comparison at P < 0.05.
Experiment 1: Effects of NaCl and PEG on the Root Conductivity and Leaf
Ion Content of Seedlings of 7 Citrus Rootstocks
The objective of this experiment was to compare the growth, ion
content, and water relations of 7 rootstocks treated with different
22
NaCI Concentration (g!_1)
2 4 6 8
Fig. 1. Osmotic potential versus NaCI concentration as
determined by vapor pressure (VPD) and
freezing point depression (FPD).
23
PEG Concentration (g L ' )
50 100 150
200
Fig. 2. Osmotic potential versus PEG concentration as
determined by vapor pressure (VPD) and
freezing point depression (FPD).
24
levels of NaCl and PEG. On October 20, 1985, 5-month-old uniform
seedlings of 7 rootstock cultivars were transplanted into 33 cm-tall
black plastic pots containing about 2.2 L of fine sand. Rootstocks
studied were the following: sour orange (Citrus aurantium), Cleopatra
mandarin (C. reshni), Swingle citrumelo (C. paradisi x Poncirus
trifoliata), Carrizo citrange (P. trifoliata x C. sinensis), rough lemon
(C. jambhiri) , Milam (C. jambhiri variant) and trifoliate orange (P.
trifoliata) . The plants were watered with a half strength Hoagland's
solution and were grown with this control solution for 2 months. Sodium
chloride and PEG treatments were started on December 19, 1985, and
nutrient solutions for treated plants were identical to that of the
control plants except for the addition of NaCl and PEG. Sodium chloride
and PEG were added to the half strength Hoagland's solution to achieve
final concentrations of -0.10, -0.20, and -0.35 MPa. The basic nutrient
solution (control) had an osmotic potential (OP) of -0.05 MPa.
Treatments were as follows:
Treatment TDS 0P_ _ EC NaCl
(mg L"1) (MPa) (d!
1. NS control : Hi Hoagl. sol. 550
2. NaCl (0.10) : 1.0 g NaCl/L Hi Hoagl. sol. 1600
3. PEG (0.10) : 55 g PEG/L Hi Hoagl. sol. 460
4. NaCl (0.20) : 2.2 g NaCl/L Hi Hoagl. sol. 3000
5. PEG (0.20) : 105 g PEG/L Hi Hoagl. sol. 400
6. NaCl (0.35) : 4.2 g NaCl/L Hi Hoagl. sol. 4900
7. PEG (0.35) : 144 g PEG/L Hi Hoagl. sol. 350
Plants were adjusted to their final NaCl and PEG concentrations
through a progression of -0.10, -0.20, and -0.35 MPa solutions at 2-day
intervals to avoid osmotic shock. Plants were then maintained at their
(MPa)
(dS ra"1)
(mmol)
-0.05
1.1
0
-0.10
3.1
17
-0.10
0.9
0
-0.20
5.4
38
-0.20
0.8
0
-0.35
8.8
72
-0.35
0.7
0
25
final osmotic levels for 5 months. The experimental unit was a single
seedling arranged in a split plot with 4 replications. The 7 salt
treatments were assigned to the main plots and the 7 rootstocks to the
subplots.
At the end of the experiment, root hydraulic properties were
evaluated while in situ on 4 seedlings of each rootstock as previously
described (Graham and Syvertsen, 1984, 1985; Levy et al., 1983;
Syvertsen and Graham, 1985). Before measuring, the soil was wetted to
field capacity to minimize possible differences in soil hydraulic
conductivity and equilibrated to 25°C in the laboratory. Each pot and
intact plant were placed in a pressure chamber. The stem was then cut
10 cm above the soil and the chamber was sealed around the cut stem.
The pressure within the chamber was increased gradually to a constant
value of 0.5 MPa. After an initial equilibration time of 10 minutes,
the weight of the liquid r xuded from the cut end was measured at least 5
times at 1 minute intervals. Osmotic potential of the exudate was
measured by a Wescor vapor pressure osmometer calibrated with NaCl
solutions.
Each root system was washed free of soil, and the total length of
fibrous roots of each plant was determined by the line-intersect method
(Tennant, 1975). Water flow per root system measured in this way
included a soil conductivity component and was expressed as weight of
exudate per unit time and pressure (ug s~ MPa~ ). Root conductivity
for each rootstock was calculated by dividing the water flow by the
total fibrous root length. Thus, the root conductivity was expressed in
ug/s/MPa per meter of fibrous roots (ug m s MPa ).
Prior to measuring root conductivity, the trunk circumference of
each seedling was measured at a point 5 cm above the soil surface and
26
converted to stem cross sectional area. Dry weights of leaves, stems,
fibrous roots, and tap roots were determined. Shoot root ratio and
specific root weight (root weight per unit length) were calculated.
Leaf chloride content was measured using a Buchler-Cotlove chloridometer
after extracting the leaf samples with a nitric-acetic acid solution.
Measurement of leaf Na, Ca, Mg, K, P, Zn, Mn, Cu, and Fe content was
performed using an inductively coupled argon plasma spectrophotometer
after a wet digestion of the samples in a nitric-perchloric acid
mixture.
Experiment 2: Water Relations of Sour Orange and Cleopatra Mandarin
Seedlings under NaCl and PEG Stresses
The objective of this experiment was to study the effects of NaCl
and PEG on the root conductivity, plant growth, stomatal conductance,
and chlorophyll content of seedlings of 2 rootstocks differing in
chloride accumulation characteristics, sour orange and Cleopatra
mandarin (Cooper et al., 1951).
Six-month-old uniform seedlings of sour orange and Cleopatra
mandarin were transplanted on November 13, 1985, into 19-cm tall black
plastic pots containing 5.5 L of Astatula fine sand. Plants were then
watered to excess every 2 to 3 days with half strength Hoagland's
solution for one month before NaCl and PEG treatments were started. The
treatments were the same as in Experiment 1. The treatments were
replicated 7 times in a split plot design with 2 main plots (rootstocks)
and 7 subplots (solutions). All variables measured in Experiment 1 with
the exception of the chemical analysis were also measured similarly in
this experiment. Seedling height from the soil surface to the terminal
bud was measured every 2 weeks. Leaf conductance to water vapor was
measured on abaxial leaf surfaces with a Li-cor 1600 steady state
27
porometer at 2-hour intervals from 0700 to 1700 hours for 3 consecutive
days.
After 5 months of NaCl and PEG treatments, two 1-cm diameter disks
were removed from the central area of 2 mature leaves per seedling to
determine leaf chlorophyll content using N, N-dimethyl formamide as a
solvent (Moran and Porath, 1980; Syvertsen and Smith, 1984). Two
millimeters of N, N-dimethyl formamide were placed in a small bottle and
the 2 leaf disks which were removed from the same seedling were weighed
and then immersed in the solvent. The bottles were firmly closed and
stored in the dark in a freezer for 2 months. The bottles were then
removed from the freezer and left in the dark to equilibrate to the
temperature of the laboratory prior to spectrophotometer examination.
One millimeter of the chlorophyll extract from each bottle was diluted
with deionized water and examined by means of a scanning
spectrophotometer. The optical density of the extract was measured at
wavelengths of both 663 mu and 644 mu, and chlorophyll content was
calculated following the equations used by Arnon (1949). Leaf
chlorophyll content was expressed as mg of leaf chlorophyll per gram
fresh weight.
New shoot growth was determined by counting leaf number and
measuring leaf area over a 3-month period. The plants were harvested
after 6 months of NaCl and PEG treatments. Total leaf area was measured
by a Li-cor leaf area meter. Fresh and dry weights of leaves were
determined. Specific leaf weight (SLV), expressed on a fresh and dry
weight basis per unit of leaf area, was calculated. Leaf succulence was
expressed as grams of water per gram of leaf dry weight.
28
Experiment 3; Fibrous Root Density and Distribution of Sour Orange
Seedlings under NaCl and PEG Stresses
The objective of this experiment was to determine the effect of NaCl
and PEG on the root growth and distribution of sour orange seedlings.
Five-month-old seedlings were transplanted on October 1, 1985, into root
boxes filled with Astatula fine sand. The root boxes were similar to
those described by Bevington and Castle (1982, 1985). Each container
consisted of one plexiglas sheet (6.4 mm thick) attached to the front of
a wooden box. The plexiglas was covered with a removable metal shutter
to exclude light. The internal dimensions of a root box were 87 cm
high, 27 cm wide, and 5 cm thick. The viewing surface was 23 dm and
the volume was about 11.5 L. Drainage was provided by 3 mesh-covered
outlets in the bottom of the box. The boxes were vertically oriented.
Seedlings were allowed to adjust in their containers for 2 months.
During this period, they were watered every other day with half strength
Hoagland's solution. Plants were then treated with 2 concentrations of
NaCl and PEG (total osmotic potential equal to -0.12 and -0.24 MPa).
The experimental design was a randomized complete block with 3
replications using a single seedling per box. Treatments were as
follows:
Treatment TDS OP EC NaCl
1. NS control
2. NaCl (0.12)
3. PEG (0.12)
4. NaCl (0.24)
5. PEG (0.24)
(mg L ) (MPa) (dS m ) (mmol)
% Hoagland's sol. 550 -0.05 1.1 0
1.1 g NaCl/L ¥2 Hoagl. sol. 1700 -0.12 3.3 19
60 g PEG/L V2 Hoagl. sol. 450 -0.12 0.9 0
2.8 g NaCl/L V? Hoagl. sol. 3300 -0.24 5.9 48
110 g PEG/L 'k Hoagl. sol. 390 -0.24 0.8 0
29
Root growth was recorded at 2-week intervals by using colored
pencils to trace the root system onto transparent acetate sheets. Plant
height was measured at 2-week intervals. Stomatal conductance was
measured about every 2 weeks and for 2 consecutive days at 2-hour
intervals from 0700 to 1700. After 6 months of NaCl and PEG treatments,
the plants were taken from their boxes by removing the plexiglas wall
and inserting a needle board to hold the root system in place. Leaves,
stems, and roots were separated and roots were divided in place into 3
equal compartments (top, middle, and bottom). Shoot and root dry
weight, shoot root ratio, leaf number, plant height, root length,
specific root weight, and stomatal conductance were determined as
described in Experiment 2.
Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity
The objective of this experiment was to determine and quantify the
growth and water relations of sour orange seedlings when only a portion
of the root system was exposed to NaCl or PEG. A split-root system was
initiated using the technique of Koch and Johnson (1984). The tap root
of each seedling at the 3-leaf stage was cut to a 1 cm length and all
other roots were removed. The remaining portion of the tap root was
dipped into a 50% ethanol solution containing 5 grams of IBA
(indolebutyric acid) per liter. Seedlings were then placed in PR0MIX
BX, watered daily and fertilized weekly for 2 months. Seedlings which
had 2 uniform adventitious root systems were selected and transplanted
when 5 months old into 2.2 L square plastic containers stapled together
along one side (Fig. 3). These seedlings were left to adjust in their
double pots for 1 month before NaCl and PEG treatments were imposed.
The treatments were replicated 4 times in a randomized complete block
design and are shown below:
30
Fig. 3. Sour orange seedlings with a split-root system.
a. Root development after 2 months.
b. Container system used to grow split-root seedlings.
Treatment
1. NS/NS (no salt)
2. NS/NaCl (0.10)
3. NaCl (0.10)/NaCl (0.10)
4. NS/NaCl (0.20)
5. NaCl (0.20)/NaCl (0.20)
6. NS/NaCl (0.35)
7. NaCl (0.35)/NaCl (0.35)
8. NS/PEG (0.20)
9. PEG (0.20)/PEG (0.20)
31
TDS
(mg L"1)
550/550
550/1600
1600/1600
550/3000
3000/3000
550/4900
4900/4900
550/400
OP
(MPa)
-0.05/-0.05
-0.05/-0.10
-0.10/-0.10
-0.05/-0.20
-0.20/-0.20
-0.05/-0.35
-0.35/-0.35
-0.05/-0.20
-0.20/-0.20
EC
(dS m"1)
L. 1/1.1
1.1/3.1
3.1/3.1
1.1/5.4
5.4/5.4
1.1/8.8
8.8/8.8
1.1/0.7
0.7/0.7
400/400
Water relations variables were monitored on 4 successive days during
the fourth month of salt treatment. Leaf water potential was measured
at sunrise and at midday on fully expanded leaves using a pressure
chamber. Leaves were then removed from the chamber, trapped in double
plastic bags and rapidly frozen at -20°C. Leaves were subsequently
thawed after 48 hours and their osmotic potential was determined with a
vapor pressure osmometer. Turgor potential was obtained by subtracting
the osmotic potential value from the water potential value. Morning and
midday stomatal conductance and leaf transpiration rates were measured
with a steady state porometer. For anatomical study, 2 mature leaves
per plant from NS/NS and NaCl (0.35)/NaCl (0.35) treatments were
selected from about half-way between the first leaf and the shoot apex.
Two small rectangles were cut at mid-lamina of each leaf, frozen
immediately, and cut by a Cryostat minot rotary microtome in sections 10
microns thick. Sections were then thawed in a phosphate buffer saline
solution. Twenty randomly selected leaf cross sections per treatment
were fixed for a light microscopy study.
32
After 4 months of NaCl and PEG treatment, the plants were harvested,
and shoot and root dry weights were determined.
Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under
Saline Conditions
The objective of this experiment was to determine if the addition of
calcium to saline irrigation water would reduce salt damage.
Three-month-old sour orange seedlings were transplanted on August 10,
1986, into the same pots used in Experiment 2. Salt treatments
(Table 1) were started after 1 month of adjustment, and seedlings were
irrigated every 2 to 3 days for 4 months. The treatments were
replicated 8 times in a randomized complete block design. The plants
were watered the night before harvest and leaves were removed the
following morning. Fresh and dry weights of leaves, stems, and roots
were recorded. The succulence of new and old leaves was computed. The
dried, mature, fully expanded leaves were ground and their mineral
content was determined as in Experiment 1.
33
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RESULTS
Experiment 1; Effects of NaCl and PEG on the Root Conductivity and Leaf
Ion Content of Seedlings of 7 Citrus Rootstocks
The results of the analysis of variance showed that salt treatments
and rootstocks were significant and independent factors; i.e., the
interaction of these 2 factors was not significant.
Significant differences in growth due to NaCl and PEG treatments
were found among rootstocks. Shoot dry weight generally decreased as
NaCl and PEG concentration increased in the nutrient solution (Table 2).
Shoot dry weight at the low, medium, and high NaCl concentrations was 18
to 36%, 30 to 55%, and 58 to 82% lower, respectively, than the control
plants. Shoot dry weight of sour orange (SO) and Cleopatra mandarin
(CM) seedlings was the least affected while Milam (ML) and Poncirus
trifoliata (PT) seedlings showed the greatest response. Sodium chloride
and PEG effects on root dry weight were similar to those on shoot dry
weight (Table 3). However, roots were less affected than shoots so that
the shoot-root ratio decreased with increasing NaCl and PEG
concentration (Table 23, Apendix). Total plant dry weight (Table 24,
Appendix) and stem cross sectional area (Table 25, Appendix) were
proportionally reduced by NaCl and PEG concentrations and reductions
were usually greater with PEG than with NaCl. Fibrous root length was
also reduced by NaCl (Fig. 4) but specific root weight (SRW, dry weight
per unit length) increased with increasing NaCl concentration (Table 4).
34
35
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co a.
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37
# 80
10
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u.
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o 80
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Q
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Ik
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so
CM
RL
r-.CC ML
nifhiimihny
cc
t>c
sc
PT
CM
! I -
"I SO n
ML
RL
Rootstock
Fig. 4. Effect of 3 NaCl concentrations (a = -0.10 MPa,
b = -0.20 MPa, c = -0.35 MPa) on the total
fibrous root length, root hydraulic conductivity,
and water flow rate for seedlings of 7 citrus
rootstocks.
6 *->
C
bO <D
e u
^ 01
38
(0
M
3
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u
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39
The increase in SRW of rough lemon (RL), ML, and PT was greater than
that of the other rootstocks.
Root hydraulic variables were affected by rootstock and NaCl.
Significant differences in root conductivity among rootstocks were found
under non-stresed conditions (Table 5) as well as under NaCl stress
conditions (Fig. 4). Sour orange and CM had the smallest reduction in
hydraulic conductivity and ML and PT had the greatest. There was a
significant negative relationship between root hydraulic conductivity
and SRW of the 7 rootstocks studied (Fig. 5). As root weight per unit
length increased, conductivity decreased.
Water flow through the root system decreased as much as 41 to 89%
at the first NaCl level (Fig. 4). Osmotic potential of root exudate due
to NaCl stress followed the same trend as root hydraulic conductivity
(Fig. 20, Appendix). Water flow and osmotic potential of root exudate
were reduced the least in SO and CM and the most in ML and PT. However,
when NaCl was not added to the irrigation water, PT and Swingle
citrumelo (SC) had the highest osmotic potential of root exudate, and SO
and CM had the lowest potentials (Table 5).
Leaf burn symptoms appeared in the NaCl (0.35) treatment in PT and
ML after 5 weeks. In RL, SC, Carrizo citrange (CC), and SO, leaf burn
symptoms occurred after 6 weeks at the highest NaCl concentration (-0.35
MPa). Just before harvest, final evaluation of the different rootstocks
based on tree appearance and performance was made (Table 6, Fig. 6).
Leaf ion content of the seedlings of the 7 rootstocks was affected
by the NaCl and PEG concentrations. Sodium (Table 7) and chloride
(Table 8) contents in the leaves of all rootstocks increased with
increasing NaCl in the nutrient solution. Cleopatra mandarin
accumulated the least chloride while PT, SC, and CC accumulated the
40
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41
50 60 70
Specific Root Wt (mg m 1 )
Fig. 5. Relationship between root hydraulic conductivity
and specific root weight of seedlings of 7 citrus
rootstocks under non-stressed conditions.
42
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43
Fig. 6. Effect of NaCl at an osmotic potential of -0.35 MPa on
the 7 rootstocks after 5 months of salinity treatments.
44
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46
least sodium. Even at a relatively low NaCl concentration (-0.10 MPa),
large amounts of chloride were accumulated in SO, RL, ML, and PT leaves.
Large amounts of sodium were also accumulated in RL and ML leaves. The
accumulation or exclusion characteristics of sodium and chloride for
each rootstock are summarized in Table 9.
Sodium chloride at -0.35 MPa reduced leaf calcium of all
rootstocks 10 to 40% with the exception of PT while PEG generally
increased calcium content (Table 10). Both NaCl and PEG reduced
magnesium (Table 26, Appendix). Magnesium reduction varied among
rootstocks and ranged from 28 to 50% and from 22 to 41% under NaCl and
PEG, respectively.
Potassium decreased significantly in SO, CM, RL, and ML but did not
in SC, CC, and PT with NaCl treatments (Table 27, Appendix). Potassium
seemed to be more strongly reduced in PEG treatments than in NaCl
treatments.
Both NaCl and PEG had similar effects on leaf phosphorus content
but the effect was more pronounced with PEG (Table 28, Appendix).
Sodium chloride and PEG significantly increased phosphorus in CM, SC,
CC, RL, and ML, reduced phosphorus in PT, and did not affect phosphorus
in SO.
Both zinc and manganese were significantly increased under PEG
stress. In some rootstocks, PEG more than doubled the zinc and
manganese levels. Zinc was reduced in SC, CC, RL, ML, and PT but was
not in SO and CM under NaCl stress (Table 29, Appendix). Manganese
tended to increase in the leaves of NaCl-treated plants except for RL
(Table 30, Appendix).
n)
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3
-H
l-i
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48
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49
Experiment 2: Water Relations of Sour Orange and Cleopatra Mandarin
Seedlings under NaCl and PEG Stresses
As in Experiment 1, the results of the analysis of variance showed
significant differences among salt treatments and between rootstocks but
there were no significant interactions between these 2 factors.
The growth rate of SO and CM seedlings was significantly reduced
with increasing NaCl and PEG concentrations in the nutrient solution. A
NaCl concentration as low as -0.10 MPa (1600 mg L~ ) reduced shoot and
root dry weight, root length, and stem cross sectional area by 50% after
6 months of treatment (data not presented). For both rootstocks,
seedling height was 26 to 39% and 33 to 50% lower, respectively, at the
first 2 NaCl concentrations (Table 31, Appendix). Total leaf area was
reduced by more than 40% at the -0.10 MPa NaCl level (Table 32,
Appendix). All these growth variables were more severely reduced under
PEG than under NaCl stress.
No significant difference in growth reduction was found between SO
and CM. Similar to Experiment 1, shoot root ratio decreased and SRW
increased with increasing NaCl and particularly PEG in the nutrient
solution (data not shown).
Sodium chloride reduced new shoot growth of SO (Tables 11, 12).
Leaf size of new shoots was smaller for salt-treated plants than for
control plants (Table 11). Sodium chloride-treated plants had 59 to 86%
fewer leaves than those grown without salt (Table 12).
Root hydraulic conductivity and water flow of the 2 rootstocks were
reduced at the first salinity level by about 50% and more than 70%,
respectively. Water flow through the root system to the shoot in the
PEG treatment was reduced by more than 95% (data not shown). Similar to
Experiment 1, no significant differences in root conductivity, water
u
X
bOW
1
1
3
bn
rH
c
U-l
•1-1
rH
> -o
<u
(I)
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50
CO i-H rH
bo
c
m
OS
51
bO
C
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w
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G
JZ
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D
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rH
a.
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c c cu
(0 (0 >
QJ <D <V
ac ac i-i
52
flow and osmotic potential of root exudate were found between SO and CM.
There was a positive correlation between water flow through the root
system and osmotic potential of root exudate (Fig. 7). With Nad, less
water flow corresponded to higher ion concentrations in the root exudate
and consequently to a lower osmotic potential of the root exudate. Both
NaCl and PEG increased SLW when expressed on a dry weight basis (Table
33, Appendix). However, unlike NaCl, PEG decreased SLW when expressed
on a fresh weight basis. Consequently, leaf succulence was decreased by
PEG and increased by NaCl (Table 13). Among PEG treatments, succulence
was more reduced in SO than in CM seedlings.
Leaf chlorophyll content was reduced by NaCl and PEG treatments.
A significant difference in chlorophyll content due to NaCl was found
between SO and CM with a greater reduction occurring in SO (Table 14).
Polyethylene glycol generally reduced chlorophyll level in CM more than
did NaCl.
Stomatal conductance was also affected by NaCl (Figs. 21, 22,
Appendix) and PEG (Figs. 23, 24, Appendix). No significant difference
in stomatal conductance was found between SO and CM under NaCl and PEG
stresses. Again, the effect of PEG was more pronounced on this variable
than that of NaCl. There was a significant positive linear correlation
between root hydraulic conductivity and midday stomatal conductance
(Fig. 8).
Addition of NaCl and PEG to the nutrient solution reduced seedling
water use or evapotranspi ration. Water use could be approximated
because the amount of water added each time was based on bringing the
soil to slightly more than field capacity. Estimated water use for NaCl
(0.10), NaCl (0.20), NaCl (0.35), PEG (0.10), PEG (0.20), and PEG (0.35)
53
■0.1 -0.2 -0.3
Salt Treatment (MPa)
Fig. 7. Relationship between water flow rate and
osmotic potential of root exudate of sour
orange and Cleopatra mandarin seedlings.
54
Table 13. Leaf succulence [(g water/g dry wt) x 100] of
seedlings of 2 rootstocks grown for 6
different NaCl and PEG concentrations-
months under
-Experiment 2.
Sour
orange
Cleopa
tra mandarin
Treatment
X difference
X difference
(-MPa)
Mean1
than NS
Mean
than NS
NS control
179
by
0
125 b
0
NaCl (0.10)
190
ab
+6
125 b
0
NaCl (0.20)
191
ab
+ 7
125 b
0
NaCl (0.35)
194
a
+8
144 a
+15
PEG (0.10)
63
c
-65
81 c
-35
PEG (0.20)
51
c
-72
50 d
-60
PEG (0.35)
27
d
-85
52 d
-58
rMean of 7 plants.
yMean separation within columns by Duncan's Multiple Range Test,
0.05 level.
55
Table 14. Total chlorophyll (mg g~ fresh wt) of seedlings
of 2 rootstocks grown for 6 months under different
NaCl and PEG concentrations — Experiment 2.
Treatment
(-MPa)
Sour
orange
X lower
Mean2
than NS
1.99 ay
0
0.88 b
56
0.61 c
69
0.59 c
70
0.88 b
56
0.58 c
71
0.56 c
72
Cleopatra mandarin
% lower
Mean than NS
NS control
NaCl (0.10)
NaCl (0.20)
NaCl (0.35)
PEG (0.10)
PEG (0.20)
PEG (0.35)
2. 42 a
0
2.15 a
11
1.64 b
32
1.12 c
54
1.20 c
50
0.95 c
61
0.83 c
66
:Mean of 7 plants.
Mean separation within columns by Duncan's Multiple Range
Test, 0.05 level.
56
E
u
<D
O
c
■*—
o
"O
c
o
O
TO
E
o
CO
.30 _
Root Conductivity (ug s~ m MPa )
Fig. 8. Relationship between midday stomatal
conductance and root conductivity of sour
orange and Cleopatra mandarin seedlings.
57
treatments were, respectively, 50, 25, 17, 25, 12, and 12% of that for
the control (NS) treatment.
Experiment 3: Fibrous Root Density and Distribution of Sour Orange
Seedlings under NaCl and PEG Stresses
Plant responses in this experiment to NaCl and PEG treatments were
similar to those obtained in Experiment 2. Shoot and root dry weight,
shoot root ratio, and leaf number generally decreased with increasing
NaCl and PEG concentrations in the nutrient solution (data not shown).
Significant differences among treatments were found in stomatal
conductance during different months (Fig. 9) as well as in daily
stomatal conductance (Fig. 10). Stomatal conductance also decreased as
leaf age increased (Fig. 9). Throughout the growing period, shoot and
root growth rate increased with time, but the growth rate of stressed
seedlings was less than that of non-stressed seedlings. After 4 weeks,
measurements of seedling height (Fig. 11) and root length (Fig. 12)
showed a significant reduction in plant growth due to NaCl and PEG
treatments. Cycling between shoot and root growth was noticed under
stressed and non-stressed conditions (Fig. 13).
When the portion of the root system in each compartment (top,
middle, and bottom) of the root box was compared, root density decreased
with depth and was significantly higher in the top compartment than in
either of the lower 2 sections (Table 15, Fig. 14). Seedlings receiving
NaCl or PEG treatments developed a shallow root system as compared to
the control (Fig. 14). Stressed seedlings had a higher percentage of
the total root system in the top of the root boxes. About 65% of the
roots of the PEG-stressed seedlings, but less than 50% of the roots of
control seedlings, were located in the upper section (Table 15). In the
58
Sour orange
E 60
.50
.40
2 .30
E
o
w .201-
10-
Dec. 4 Feb. 1 1
TIME (date)
NS
• NaCI(12)
■ NaCI(24)
oPEG(. 12)
°PEG(.24)
Apr. 17
Fig. 9. Midday stomatal conductance of sour orange seedlings
irrigated with nutrient solution containing no salt
(NS) or with added NaCl or PEG.
59
E
o
d>
o
c
TO
O
"O
c
o
o
ro
E
o
CO
Sour orange
20
10
\\NS
x\NaCI(12)
^ ■NaCK.24)
A ^°^0PEG(.12)
'^.u— d— d— d P E G( . 2 4 )
11
15
*t
1 1
15
Time ( hr)
Fig. 10. Relationship of time of day to stomatal conductance of
sour orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCl or PEG
during 2 consecutive days. Measurements were started
on April 17, 1986. Seedlings were irrigated the day
before measurements were started and not irrigated
until after measurements were completed on Day 2.
60
60
50 -
o 40
en
c5 30
- 20 -
"O
CD
CD
en
10 -
Sour orange
^ NS
. NaCK. 12)
. NaCK. 24)
/ /-^"
^l PEG(12)
^ Jk>^
^^ PEG(.24)
1 1
•
Dec 4
Mar 6
TIME (date)
Jul. 1 7
Fig. 11. Growth of sour orange seedlings irrigated with
nutrient solution containing no salt (NS) or
with added NaCl or PEG.
61
Sour orange
O)
c
Z 2
o
NS
• NaCI(.12)
■ NaCK.24)
o PEG(12)
a PEG(24)
Dec. 4
Mar.6
TIME (date)
Jul. 1 7
Fig. 12. Fibrous root length of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or with
added NaCl or PEG.
62
5
o
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10
6 -
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30
2 20
CD
o
o
a:
10
NaCK.12)
NaCK.24)
0 PEGU 2)
0 PEG(.24)
Dec Jan
Feb Mar Apr
TIME (month)
May
Fig. 13. Fluctuations in shoot and root growth of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCl or PEG.
63
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Fig. 14. Root density and distribution of sour orange seedlings
growing in root boxes under non-stressed (NS) and
stressed (NaCl, PEG) conditions. NaCl and PEG
treatments were at -0.24 MPa osmotic potential.
65
bottom section, only 5 to 16% of the roots developed in the stressed
chambers as compared to 26% in the controls.
Fibrous root length at the plexiglas face, measured from tracings
made on acetate sheets with colored pencils, was compared to the total
fibrous root length measured at the end of the experiment. Root length
against the plexiglas represented 3 to 4%, 2 to 3%, and 4 to 5% of the
total root length in the top, middle, and bottom of the root boxes,
respectively. From the comparison of root lengths at the plexiglas and
in the box, it was concluded that growth and distribution of citrus
roots at the plexiglas-soil interface correlated satisfactorily with
growth and distribution of roots in the bulk soil.
Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity
Uniform salinity was significantly more damaging to sour orange
seedlings than non-uniform salinity (Table 16; Fig. 15). Shoot dry
weight was reduced only slightly (9 to 21%) when half of the root ystem
was irrigated with saline solutions. When both halves of the root
system were irrigated with saline solutions, shoot dry weight was
reduced 45 to 81% (Table 16). The trend was similar with root dry
weight in that stressing one-half of the root system resulted in only a
moderate reduction (16 to 31%) in root dry weight. Stressing both
halves gave a much larger reduction in root dry weight (43 to 79%).
In the split-root test, shoot growth did not correlate well with
the average salt stress of the total root system. The average osmotic
potential of the NS/NaCl (0.20) treatment was -0.12 MPa. Even though
this was slightly greater than the average osmotic potential of the NaCl
(0.10)/NaCl (0.10) treatment, shoot dry weight was 35% (10.7 g) less in
the NaCl (0.10)/NaCl (0.10) treatment. Similarly, shoot dry weight in
the NaCl (0.20)/NaCl (0.20) treatment was 50% (14.5 g) less than that in
66
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67
NS/PEG(.20) PEG(.20)
Fig. 15. Split-root treatment of sour orange seedlings under
uniform and non-uniform NaCl and PEG stresses. NaCl
treatments were at -0.10, -0.20, and -0.35 MPa osmotic
potentials. PEG treatments were at -0.20 MPa osmotic
potential.
68
the NS/NaCl (0.35) treatment, even though both of these treatments had
the same average NaCl stress (-0.20 MPa).
Under uniform salinity similar to Experiments 1, 2, and 3, shoot
growth was more reduced than root growth. However, under non-uniform
salinity, root dry weight on a percentage basis appeared to be more
reduced than shoot dry weight (Table 16).
Partial leaf burn occurred after 4 weeks in the NaCl (0.35)/NaCl
(0.35) treatment and after 5 weeks in the NaCl (0.20)/NaCl (0.20)
treatment. No leaf damage symptoms were noticed in the remaining
treatments until the end of the experiment.
Water relations variables were monitored on 4 successive days
during the fourth month of salt treatment. Data were combined because
no significant differences were found from day to day. Similar to
growth, water relations variables were also significantly more disturbed
under uniform salinity than under non-uniform salinity conditions. With
uniform salinity, leaf water and turgor potentials decreased
significantly from morning to midday, but leaf osmotic potential did not
(Fig. 16). Leaf water potential, osmotic potential, stomatal
conductance, and transpiration decreased with increasing NaCl and PEG
concentrations in the irrigation water (Tables 17, 18). Turgor
potential significantly increased in response to NaCl treatments
particularly during the morning. A significant positive correlation was
found between stomatal conductance and transpiration (Fig. 17). Similar
to findings of the preceding experiments, PEG at -0.20 MPa was more
damaging than NaCl at the same osmotic potential.
Cross sections of leaves from control (NS/NS) and from NaCl
(0.35)/NaCl (0.35) treatments, compared by light microscopy, showed that
the number of cell layers in the epidermis, the palisade, and the spongy
69
03
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5
1.5
u 1.0
o
■5 °-5^
Midday ~_
-0.1 -0.2 -0.3
Salt Treatment (MPa)
Fig. 16. Leaf water, osmotic, and turgor potential of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with NaCl added to
both root halves. Solid figures are morning
values and open figures are midday values.
70
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Stomatal Conductance (cm s ' )
Fig. 17. Relationship between transpiration and stomatal
conductance of sour orange seedlings.
73
raesophyll in control leaves and NaCl-treated leaves were similar.
Epidermal and palisade cells of the control and NaCl-grown leaves were
also similar in size; however, the spongy mesophyll cells of the •
NaCl-treated leaves were about 3 times larger than those of the control
(Fig. 18). The overall increase in leaf thickness due to NaCl was
relatively small (23%) because the enlarged cells of the spongy
mesophyll were tightly packed with much less intercellular space. Cells
of the spongy mesophyll in NaCl-treated leaves also had fewer
chloroplasts than those in the control leaves.
Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under
Saline ConditionI
Addition of NaCl to half strength Hoagland's solution significantly
reduced growth of sour orange seedlings. Shoot, root, and total plant
dry weights were reduced by about 30% (treatments 2 and 10) when 40 mM
NaCl was added to the nutrient solution (Tables 19, 20). However,
addition of 7.5 mM CaS04 (treatment 3) to the salty solution decreased
the adverse effect of NaCl on growth. Furthermore, addition of only
5 mM CaSO (treatment 12) completely inhibited the adverse effect of
NaCl. Addition of either KCl (treatments 6 and 7) or CaCl2 (treatments
5 and 8) to the salty solution did not improve plant growth.
In the leaves of the sour orange seedlings, addition of NaCl to the
nutrient solution significantly increased sodium and chloride, decreased
calcium, magnesium, and potassium but had little or no effect on
phosphorus, zinc, manganese, copper, and iron (Table 21). Sodium and
chloride accumulation in the leaves usually reduces growth. Addition of
CaS04 (treatments 3, 4, 11, and 12) to the saline solution reduced
sodium and chloride content and, therefore, improved plant growth.
Addition of KCl (treatment 6) did not reduce sodium and chloride; hence,
74
Fig. 18. Cross sections of sour orange leaves.
a. Leaf cross section of non-stressed seedling.
b. Leaf cross section of NaCl-stressed seedling,
i.s. = intercellular space.
75
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79
growth was not improved. Addition of CaCl2 (treatments 5 and 8) reduced
sodium but did not reduce chloride sufficiently to improve growth.
Significant growth reduction occurred without any visible symptoms
of salt damage. Although total plant dry weight was reduced by more
than 28% in some treatments after 4 months of salinity stress, none of
these treatments caused any apparent leaf damage symptoms.
Comparison of Citrus Seedling Responses to NaCl and PEG Treatments
The effects of NaCl and PEG on citrus seedlings differed in the
degree and the type of damage. When considering NaCl and PEG at similar
osmotic potentials, the damaging effects on all measured variables
generally appeared to be larger in the PEG treatment than in the NaCl
treatment. Citrus seedling responses to NaCl and PEG compared to the no
salt control are summarized in Table 22. The higher salinity damage
occurring in Experiment 2 in comparison to Experiment 1 was thought to
be mainly due to the more rapid onset of salt treatment and to the
longer duration of salt treatment.
Differences in damage and leaf burn symptoms were also found
between NaCl and PEG. Leaves from NaCl-treated seedlings appeared
abnormally thickened. Leaf symptoms in the NaCl treatment were
initially similar to nitrogen dificiency (uniform loss of the green
color over the entire leaf). Later, leaf burn occurred as large spots
merged together. Leaf scorch and areas of dead tissue extended inward
from the margins of the leaf. Sodium chloride-damaged leaves readily
abscised and dropped as soon as visual burn symptoms appeared.
Sometimes leaves fell off before they reached this stage. Leaf symptoms
in PEG treatment first appeared similar to iron-manganese deficiency
(intervenal chlorosis). Then, leaf burn appeared at the edges and
particularly at the tip of the leaf. Later, the dead area extended
inward from the tip (Fig. 19).
80
Table 22. Summary of citrus roots tock responses to NaCl and PEG as
compared to the no salt control.
Variable
NaCl
PEG
Growth
Total fibrous root length
Leaf number
New shoot growth
Total root dry weight
Total shoot dry weight
Seedling height
Stem cross sectional area
Total leaf area
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
Water Relations
Root conductivity
Water flow rate
OP of root exudate
Stomatal conductance
Transpiration
Water use
Leaf water potential
Leaf osmotic potential
Leaf turgor potential
Leaf succulence
Leaf Mineral Analyses
Chloride
Sodium
Calcium
Magnesium
Potassium
Phosphorus
Zinc
Manganese
Copper
Iron
Other Variables
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
increase
increase
increase
increase
decrease
decrease
decrease
increase
decrease
increase
no change
no change
decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
increase
decrease
no change
no change
increase
decrease
decrease
increase
increase
increase
no change
no change
Shoot root ratio
Specific root weight
Specific leaf wt (Dry wt basis)
Specific leaf wt (Fresh wt basis)
Leaf chlorophyll
decrease
increase
increase
increase
decrease
decrease
increase
increase
decrease
decrease
81
Sour orange
Control NaCI-damaged leaves
NaCI T PEG
Control
Fig. 19. Sour orange leaves from non-stressed (control)
and stressed (NaCI, PEG) seedlings.
DISCUSSION
Leaf Ion Content and Salinity Tolerance
Rootstock Tolerance
Important differences in salt tolerance among citrus rootstocks
were demonstrated in this study. Based on various measurements of plant
growth (shoot, root, and total plant dry weight), on plant water
relations factors (root hydraulic conductivity, water flow rate, and
osmotic potential of root exudate), and on seedling appearance and
performance (leaf burn, leaf drop, and dieback), SO and CM seedlings
were the least affected while the most damage occurred in ML and PT
seedlings. Rough lemon, SC, and CC had an intermediate response. From
these results, SO as well as CM were classified as relatively tolerant
rootstocks, RL, SC, and CC were sensitive, and ML and PT were very
sensitive rootstocks to NaCl. Cooper et al. (1951) and Ream and Furr
(1976), who based their conclusions on visual leaf burn symptoms and
leaf chloride content, found that CM appeared to be more salt tolerant
than SO. The current study, which was mainly based on growth and water
relations measurements, showed that SO was as tolerant as CM. This
overall classification agreed with Cooper et al. (1951) who reported
that PT was a very salt sensitive rootstock and with others who found
that CC was a salt sensitive rootstock when compared to CM (Joolka and
Singh, 1979; Patil and Bhambota, 1978).
82
83
Ion Exclusion and Accumulation
Sour orange seemed to behave differently from the other rootstocks.
Even though SO accumulated higher amounts of sodium and chloride -than PT
and its hybrids (SC and CC) at the first salinity level (Tables 7, 8),
plant growth and physiological activities of SO were relatively
unaffected as compared with those of RL, ML, SC, CC, and PT. Rough
lemon and ML were sodium and chloride accumulators similar to SO.
However, salt damage was more severe and tree growth and appearance were
poorer in RL and ML than in SO. Since excess accumulation of both
chloride and sodium in SO leaves caused relatively minor damage to this
rootstock, SO might have the ability to partially exclude these ions
from the cytoplasm where they could inhibit metabolic functions.
Salinity studies at the cellular level could further clarify ion
exclusion and compartmentalization ability in citrus.
The citrus rootstocks tested in this study are considered to be
salt sensitive because no rootstock has the ability to exclude both
chloride and sodium. Rough lemon, ML, and SO are chloride and sodium
accumulators. Poncirus trifoliata, SC, and CC are chloride accumulators
but sodium excluders. Cleopatra mandarin is a chloride excluder but a
sodium accumulator. Furthermore, sodium exclusion capacity in PT, SC,
and CC and chloride exclusion capacity in CM are limited. This study
(Tables 7, 8) showed the inability of PT, SC, and CC to exclude sodium
and the inability of CM to exclude chloride at moderate salinity levels
(-0.2 MPa). It is suggested that in any program where plants are being
screened for salt tolerance on the basis of salt exclusion, chloride
exclusion as well as sodium exclusion should be considered because the
chloride and sodium accumulating properties of a particular species are
quite different (Grieve and Walker, 1983).
84
Leaf Ion Content and Ion Toxicity
Sodium chloride was found to reduce potassium in SO, CM, RL, and ML
but not in SC, CC, and PT leaves (Table 27, Appendix). Sour orange, CM,
RL, and ML are sodium accumulators (Table 9). Sodium accumulation in
these rootstocks might be the main factor which depressed leaf
potassium. Poncirus trifoliata, SC, and CC are sodium excluders
(Table 9). The unaffected leaf potassium in these rootstocks might be
attributed to sodium exclusion.
There was an inverse relationship between chloride ion accumulation
in the leaves and salt tolerance. Usually chloride accumulation was
associated with more damage. Since PT and its hybrids (SC and CC)
accumulated large amounts of chloride, their ability to exclude sodium
(particularly at -0.10 MPa) and to maintain adequate potassium did not
help prevent these rootstocks from showing severe growth reduction and
water relation disturbances. Furthermore, although CM accumulated
excess sodium in its leaves, growth and water relations of CM were not
as severely affected at -0.10 MPa since it was a chloride excluder.
The levels of chloride and sodium accumulation at which leaf burn
symptoms developed were found to be higher than the upper limits set by
earlier investigators. Such differences are mainly attributed to
different experimental conditions. Comparison between leaf chloride
content (Table 8) and visual symptoms (Table 6) shows that a leaf
chloride content of about 1% in SC and CC and even 1.7% in SO did not
cause any leaf burn symptoms. Similarly, when comparing leaf sodium
content (Table 7) to visual symptoms (Table 6), leaf sodium content up
to 1.9% in CM did not cause visible leaf burn. However, growth and
water relations were severely altered.
85
Leaf chloride and sodium analysis is thought to provide useful
information on toxicity limits as well as on rootstock tolerance.
Harding and Chapman (1951) recommended that a leaf chloride content
exceeding 0.252 be considered indicative of chloride toxicity.
Bernstein (1969) stated that although 0.25% might not lead to obvious
chloride toxicity symptoms, it might affect the longevity of leaves and
reduce the yield. Chapman et al. (1969) suggested that 0.30% chloride
in the dry matter was regarded as the threshold value of injury and leaf
levels over 0.75% chloride would be indicative of serious growth
retardation and yield reduction. According to Abdel-Messih et al.
(1979), sodium leaf content higher than 0.36% would be critical for
developing burn symptoms in citrus leaves. These threshold values were
lower than those found in this study because of the higher degree of
stress under field and dry climate conditions.
Importance of Calcium under Saline Conditions
Under salinity conditions, addition of calcium to irrigation waters
resulted in different responses in citrus. The present study on SO
seedlings showed that the beneficial effect of calcium depended on the
anion associated with the calcium salt. Calcium sulfate was found to be
significantly more effective than calcium chloride in reducing the
deleterious effect of NaCl on growth (Tables 19, 20). Walker and
Douglas (1983) did not observe any improvement in citrus growth by
increasing calcium chloride in the growth medium. However, the earlier
work on citrus by others showed the effectiveness of calcium sulfate,
calcium nitrate, and calcium carbonate on reducing sodium concentrations
in plant tissues, in preventing the def locculation effect of sodium and
in improving tree appearance and growth (Cooper, 1961; Harding et al.,
1958b; Jones et al., 1952). LaHaye and Epstein (1969, 1971)
86
demonstrated that an increase in calcium levels by adding either calcium
sulfate or calcium chloride protected bean plants from salt injury by
restricting sodium absorption and translocation to the leaves. Failure
in effectiveness of calcium chloride in our work might have been due to
the chloride accompanying the calcium and to the sensitivity of citrus
to chloride.
Physiological Effects of NaCl and PEG
Effect of NaCl on Root Conductivity
The present study showed that root hydraulic conductivity in citrus
seedlings was severely reduced due to NaCl stress and that root
conductivity varied significantly among rootstocks under stressed and
non-stressed conditions. Under non-stressed conditions, these results
were consistent with data obtained by others (Graham and Syvertsen,
1985; Syvertsen and Graham, 1985; Syvertsen et al., 1981). Under
salinity stress, root conductivity of different citrus rootstocks has
not been thoroughly studied.
Reduced hydraulic conductivity of roots has been attributed to
several factors. Bielorai et al. (1983) suggested that reduced water
uptake by mature citrus trees irrigated with saline water was a result
of soil solute potential reduction and to root suberization. Hayward
and Blair (1942) observed a condition resembling dormancy caused by
suberization of epidermal and root cap cells of Valencia orange
seedlings irrigated with NaCl solutions. They also noted reductions in
water uptake and development of lateral roots and root hairs as salinity
increased. Walker et al. (1984) studied the anatomy and the
ul trastructure of roots from two citrus genotypes (Rangpur lime and
Etrog citron) with different abilities for chloride exclusion. They
found that NaCl increased suberization of the hypodermis and endodermis
87
closer to the root tip. This increase in suberization associated with
the lower extension rate of the root tips might be of prime importance
in reducing root permeability and root hydraulic conductivity.
O'Leary (1974) found that the reduction in root conductivity of
beans could be reversed after a 2-day exposure to high NaCl by removing
the NaCl. This short time reversibility suggested that biochemical or
membrane changes were responsible for the reduced conductivity because
root suberization may not have yet occurred. Thus, differences in root
hydraulic conductivity may arise from anatomical and biochemical
features.
Effect of PEG on Root Conductivity
Polyethylene glycol was found to reduce root hydraulic conductivity
more severely than NaCl. Similar to NaCl, reduction in root
conductivity due to PEG was attributed to reduced root permeability.
Reduction in root permeability might result from root suberization,
inhibited root hair formation, and oxygen deficiency around the roots.
Growth of root hairs in redtop grass seedlings and in Vicia faba was
completely inhibited by a PEG concentration of -0.2 MPa (Jackson, 1962;
Zahran and Sprent, 1986). It was suggested by Mexal et al. (1975) that
the main damage of PEG to plants was caused by low oxygen solubility and
slow oxygen transport to the roots. In the present study, after the
termination of the first 4 experiments when the roots were removed and
washed from the soil, there was an indication that the root system in
PEG treatments suffered aeration deficiency since PEG-treated soil was
found to be firmer and sticky. Furthermore, under osmotic stress,
citrus roots exhibited early suberization of the endodermis and root
hairs were directly affected by soil water conditions (Cossmann, 1940).
88
Effect of NaCl on Stomatal Conductance
In all experiments, stomatal conductance was reduced significantly
with an increase in NaCl or PEG concentrations. It is apparent from
Fig. 8 that there is a strong correlation (R = 0.99) between stomatal
conductance and root conductivity. Even though there was a strong
correlation, closure of stomata might not be caused entirely by
salinity-induced water stress. This possibility was based on the data
presented in Table 17 which showed leaf turgor maintenance in salt
treated plants. Stomatal closure was similarly reported in certain
glycophytes when grown under saline conditions, even when leaf turgor
was maintained (Gale et al, 1967; Meiri and Poljakof f-Mayber, 1970).
O'Leary (1969) also found that stomatal conductance of beans grown in
salinized solutions was also lower than that of control plants. He
suggested that the increase in resistance (or decrease in conductance)
in the water flow pathway could result in the bean leaves experiencing
physiological drought even if osmotic adjustment occurred.
The work of Walker and his coworkers showed more clearly the
importance of sodium concentration in affecting stomata of citrus under
salinity stress. Stomatal recovery occurred in leaves of
stress-relieved Etrog citron (C. medica) even though the leaves retained
high chloride concentrations and low sodium concentrations (Walker et
al., 1982). There was a failure of stomatal recovery of Valencia leaves
on other citrus rootstocks which was associated with retention of high
sodium concentration (Walker et al., 1983). It is possible that high
amounts of sodium replaced potassium in the vacuoles and guard cells and
caused stomata to close (Behbondian et al., 1986).
89
Stomatal behavior in citrus under NaCl stress is mainly affected by
ion accumulation and therefore, better associated with the leaf osmotic
potential and not the bulk turgor potential of the leaves.
Effect of PEG on Stomatal Conductance
Stomatal conductance in citrus was more reduced under PEG stress
than under NaCl stress (Figs. 9, 10). These results agreed with those
of Plaut and Federman (1985) and with Sanchez-Diaz et al. (1982) who
found that PEG decreased leaf conduction and carbon dioxide fixation
rate in tomato and legume plants more severely than did NaCl. Reduction
in stomatal conductance due to PEG might be attributed to several
factors such as drought stress caused by reduced water flow to the
shoots and to translocation of PEG to the leaves. It was suggested by
Lawlor (1970) that PEG blocked the water pathway and induced desiccation
in plants. It was also concluded that PEG damage was due to its uptake
and translocation throughout the plant (Emmert, 1974; Kaufmann and
Eckard, 1971; Lagerwerff et al., 1961; Lawlor, 1970). It is possible
that PEG was absorbed and translocated in leaves of citrus seedlings
since PEG caused leaf necrosis.
Effect of NaCl and PEG on Chlorophyll
Leaf chlorophyll content was the only variable more significantly
affected in SO than in CM (Table 14). For SO seedlings, chlorophyll
reduction due to NaCl was similar to that due to PEG. However, for CM,
chlorophyll reduction was more severe under PEG than under NaCl
treatments. Since NaCl did not reduce chlorophyll as much in CM, and
since CM is a chloride excluder, chlorophyll reduction could be mainly
attributed to chloride accumulation in the leaves. Contrary to this,
Bhambota and Kanwar (1970) attributed salt induced chlorophyll reduction
in sweet orange to sodium uptake and to a reduction in magnesium and
90
iron uptake. Leaf chlorophyll content has also been found to be reduced
in many other crops such as beans (Seemann and Critchley, 1985) and
spinach (Downton et al., 1985; Robinson et al., 1983). In other -
photosynthetic related processes, NaCl was found to inhibit the Hill
reaction (Sivtsev, 1973) and increase the hydrolytic activity of
chlorophyllase (Sivtsev et al., 1973) in tomato leaves.
Effect of NaCl on Leaf Thickness and Succulence
Increases in leaf succulence and thickness have been attributed to
changes in cell size, cell layer number, or a combination of both. In
the present study, examination of leaf sections by light microscopy
(Fig. 18) suggested that an increase in spongy mesophyll cell size
rather than an increase in cell number caused the greater leaf thickness
and succulence. Similar conclusions were made with tobacco (Flowers
et al., 1986), spinach (Robinson et al., 1983), and beans (Wignarajah
et al . , 1975). However, in Atriplex, cotton, and Salicornia herbacea,
leaf thickness and succulence increased not only due to a development of
larger cells but also to an increase in cell layers of the mesophyll.
In most circumstances, increase in succulence was accompanied by an
increase in sodium and chloride concentrations in the leaves.
Growth of Citrus Rootstock Seedlings under NaCl and PEG Stresses
The results of this study are consistent with those of other
investigators who classified citrus as a salt sensitive crop. Growth
was reduced at least 20% in the rootstocks ranked as tolerant when
irrigated with a nutrient solution containing as little as 1 g NaCl/L
(-0.1 MPa). The higher salinity damage occurring in Experiment 2 in
comparison to Experiment 1 was thought to be mainly due to the more
rapid onset of salt treatment and to the longer duration of salt
treatment .
91
Relationship of Leaf Damage Symptoms to Growth Reduction
Significant growth reduction and physiological disturbances were
found to precede visible leaf symptoms. When comparing shoot dry weight
(Table 2) to visual symptoms (Table 6), growth reduction up to 30%
occurred without being accompanied by visible leaf damage symptoms.
Similar to this, the use of saline irrigation water decreased grapefruit
and orange yields from 18 to 54% without apparent toxicity symptoms
(Bielorai et al., 1978, 1983: Bingham et al., 1974; Francois and Clark,
1980). Salinity effects develop slowly so that leaf injury symptoms
appear only after a certain period of time. Leaf symptoms are,
therefore, a poor parameter for evaluating salt damage.
Root Growth and Distribution under NaCl and PEG Stresses
The present study showed that root growth of citrus was severely
reduced even at relatively low concentrations of NaCl and PEG in the
nutrient solution. The average daily root growth rate was reduced by
30 to 50% at -0.12 MPa NaCl and PEG, respectively (Table 15).
Nevertheless, these results showed that citrus roots were able to grow
slowly at an osmotic potential of -0.24 MPa. Bevington and Castle
(1985) reported that citrus root growth was significantly reduced at a
soil matric potential of -0.05 MPa and Monselise (1947) reported that
citrus root growth was very limited at soil water potentials of -0.75 to
-0.80 MPa. Within the limited range of NaCl and PEG used in the current
study, it is not possible to specify a water potential value at which
growth stopped completely.
Root distribution of stressed seedlings was altered in comparison
to root distribution of control seedlings. Stressed seedlings had a
higher percentage of the total root system in the top and a much lower
percentage at the bottom of the root boxes (Table 15). Seedlings
92
receiving NaCl or PEG treatments produced, therefore, a shallow root
system.
Root and shoot growth was found to be cyclic (2-month cycle), in
young citrus seedlings even when the plants were under NaCl or PEG
stress (Fig. 13). It was observed that immediately following the
cessation of shoot elongation, a rapid increase in root growth occurred
and continued until the initiation of the next shoot growth flush.
Alteration of root and shoot growth activity in citrus has been
described earlier under non-stressed conditions by other investigators
(Bevington and Castle, 1982, 1985; Marloth, 1949).
Effect of Non-Uniform Salinity and Water Stress
Soil water content and salinity levels are seldom uniform in the
field, particularly with the use of microsprinklers which may irrigate
only a portion of the root zone. A split-root experiment was designed
to determine if non-stressed portions of the root system compensated for
the decrease in water and nutrient uptake by the stressed portions so
that plants could withstand substantial amounts of stress.
The non-stressed roots were found to partially compensate for the
decrease in water by the stressed roots. Water uptake from each of the
2 sides was estimated since the amount of water added each time was
based on bringing the soil to slightly above field capacity. Water
uptake by the unsalinized half of the root system increased when the
other half of the root system was subjected to salinity stress. Similar
results were obtained on corn (Bingham and Garber, 1970) and alfalfa
(Shalhevet and Bernstein, 1968). Watering one part of the root system
of wheat (Lawlor, 1973) and tomato (Tan et al., 1981) resulted in a
compensatory increase in water uptake by other parts of the root system
so that plant water relations remained relatively unaffected. However,
93
compensation was not seen in beans and barley because plants with half
their roots in saline solutions had growth and water relations values
intermediate between those of plants grown in non-saline solutions and
plants grown in saline solutions (Kirkham et al., 1969, 1972). Only
partial compensation occurred in SO seedlings since plants with half
their root systems in either NaCl or PEG solutions had shoot and root
dry weight and leaf water and osmotic potential values closer to those
of the non-stressed control than to those with completely stressed root
system (Tables 16,17).
No soil water measurements were recorded in the split-root
experiment. However, the root dry weight data (Table 16) might indicate
that some water could have been transported through roots from the
non-stressed half to the stressed half. Several investigators have
demonstrated that plant roots can absorb water from a wet soil,
transport the water, and build up the moisture of a dry soil. In a
study using wheat plants with roots split between soil and nutrient
solution, Kirkham (1980) showed transport of water from the solution to
the soil suggesting that roots were acting like wicks.
While the present study was carried out under greenhouse
conditions, it provided several useful observations which are relevant
to field conditions. Citrus as a deep and dense rooted crop may
tolerate certain levels of salinity as long as a portion of the root
system remains in a relatively non-saline soil.
Comparative Effects Between NaCl and PEG
Although PEG-induced water stress is osmotic in nature and may not
be exactly the same as the water stress occurring in soils, it is a
sensitive method that can create small degrees of water stress on a
continuous basis not easily induced in soils (Gergely et al., 1980).
94
Polyethylene glycol was used satisfactorily by several investigators for
various species (Janes, 1966; Kaufmann and Eckard, 1971; Kaul, 1966) in
which the response to PEG was attributed to a decrease in osmotic,
potential with no obvious toxic effects.
Injuries to citrus rootstock seedlings by PEG were greater than the
osmotic effects per se. Damage due to ionic effects of sodium and
chloride were less than the damage from non-ionic PEG. Excess damage
might be attributed to insufficient transport of oxygen to the root
system due to high PEG viscosity and its effect on soil stickiness and
firmness. Damage could also be caused by PEG uptake and transport to
the leaves where it caused dehydration and leaf damage. Oxygen
availability could be significantly reduced at relatively low PEG
concentrations (Mexal et al., 1975). Absorption and secretion of PEG
4000 and 6000 by Solanaceae species was observed (Yaniv and Verker,
1983), as well as the appearance of white material on the upper surface
of bean leaves grown in PEG 20000 (Lagerwerff et al., 1961).
Studies with other species have shown NaCl to be either more
damaging or less damaging than PEG. Similar to results of the present
investigation, the damaging effect of PEG was found to be higher than
NaCl at equal osmotic potentials in tobacco (Heyser and Nabors, 1981)
and tomato (Plaut and Federman, 1985). However, growth of beans, maize,
and barley was substantially better with PEG than isosmotic salt
solutions (Lagerwerff and Eagle, 1961; Storey and Wyn Jones, 1978). The
matter of separating toxic ion effects from osmotic effects of salts on
citrus was not clearly determined in this study. This important
question merits continued investigation involving the testing of other
non-ionic compounds or nutrient solutions at different concentrations.
SUMMARY AND CONCLUSIONS
The conclusions from this study are summarized below:
1. Differences in sodium and chloride exclusion capacity were found
among citrus rootstocks. This study was the first to show that ML was a
chloride and sodium accumulator while SC and CC were chloride
accumulators but sodium excluders.
2. Citrus rootstocks were found to be sensitive to NaCl since none
of these rootstocks was able to exclude both chloride and sodium.
Furthermore, sodium and chloride exclusion capacities were lost at a
concentration of -0.20 MPa. Differences in NaCl sensitivity among
rootstocks were also found. Cleopatra mandarin and SO were the least
sensitive, ML and PT were the most sensitive, and RL, SC, and CC were
intermediate in sensitivity.
3. This study was also the first to show that salt tolerance in
citrus rootstocks was not strongly correlated with chloride and sodium
exclusion. Even though SO accumulated higher amounts of chloride and
sodium than PT, SC, and CC at an osmotic potential of -0.10 MPa NaCl,
growth of SO was as good as CM and significantly better than the
remaining rootstocks. Water relations measurements also showed that SO
was similar to CM but less stressed than the other rootstocks. Sour
orange and CM acted through 2 different mechanisms to tolerate salinity
stress. Cleopatra mandarin tolerated high concentrations of NaCl by
partial exclusion of chloride. Sour orange tolerated NaCl by possible
95
96
compartmentation of sodium and chloride and their exclusion from the
cytoplasm where they could inhibit metabolic processes.
4. Sodium chloride usually caused less damage than PEG to seedlings
of 7 citrus rootstocks. Both NaCl and PEG caused significant growth
depression and physiological disturbances even at a concentration of
-0.10 MPa. The response of citrus rootstocks to the 2 compounds was
different suggesting that NaCl and PEG acted through different
mechanisms. Addition of PEG to the irrigation water probably reduced
aeration and also moved to the shoots where it caused plant dehydration
and leaf damage. Addition of NaCl reduced water uptake but increased
leaf sodium and chloride concentrations. This increased leaf thickness
and succulence and caused leaf burn.
5. Growth reduction and physiological disturbances were found to
precede visible damage. Growth was reduced up to 30% without being
accompanied by visible leaf injury symptoms. Leaf burn symptoms
developed only after a threshold value of chloride accumulation (1%) was
reached. Leaf burn symptoms developed too slowly to accurately evaluate
salt damage.
6. In all NaCl and PEG treatments, growth was depressed and water
balance was disturbed. Growth depression was shown by plants that had
lower dry weight, fewer leaves per plant, smaller area of individual
leaves, shorter height, smaller stem cross sectional area, and smaller
root systems. Disturbance in water balance was shown by reductions in
root hydraulic conductivity, stomatal conductance, transpiration, water
use, and leaf water and osmotic potentials.
7. Root observation boxes were used to follow root growth and
distribution under stressed and non-stressed conditions. Root growth
usually alternated with shoot growth but this alternating pattern was
97
not shifted by NaCl and PEG stresses. However, root growth and
distribution were significantly affected. Seedlings receiving NaCl or
PEG treatments, produced smaller and shallower root systems with .the
majority of the roots occurring in the top layer.
8. Although roots were in direct contact with NaCl and PEG
solutions, shoot growth was more reduced than root growth. The monthly
or daily amount of new flush area was found to be a sensitive measure of
the effects of NaCl on plants. Root conductivity correlated better with
salinity tolerance among rootstocks than did total fibrous root length.
9. Stomatal conductance was greatly reduced even though leaf turgor
was maintained. This reduction was attributed to a decrease in water
flow to the shoots and to a suggested lack of osmotic adjustment of the
guard cells. Reduction in stomatal conductance correlated with an
accumulation of sodium and chloride in the leaves and depressed
transpiration and water use. Reduction in water consumption of the
stressed seedlings was attributed to lower transpiration rate per leaf
and also to smaller total transpiring area per plant.
10. Examination of leaf sections by light microscopy suggested that
an increase in cell size rather than cell number was responsible for the
increased leaf thickness. Leaf succulence and thickness increased due
to the development of larger cells in the spongy mesophyll in response
to ion accumulation. Microscopic examination also showed a decrease in
leaf intercellular space and the number of chloroplasts under NaCl
stress. Leaf chlorophyll content decreased in salt-treated seedlings
which agreed with microscopic observations. Chlorophyll reduction was
mainly attributed to chloride accumulation in the leaves.
11. The split-root experiment demonstrated that citrus shoot growth
did not correlate with the average osmotic potential of the 2 root
98
halves. It demonstrated also that citrus could withstand substantial
amounts of stress as long as half of the root system was growing in a
non-stressed environment.
12. Sodium chloride reduced calcium and magnesium contents but
increased phosphorus content in the leaves. Sodium chloride decreased
leaf potassium content only in sodium-accumulator rootstocks (SO, CM,
RL, and ML).
13. This study showed that the beneficial effect of adding calcium
to saline irrigation water depended on the anion accompanying the salt.
Calcium sulfate but not calcium chloride was found to overcome the
detrimental effects of NaCl by decreasing the concentrations of sodium
and chloride in the leaves.
APPENDIX
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108
Table 31. Seedling height (cm) of seedlings of 2 rootstocks
grown for 6 months under d
concentrations — Experiment
Lfferent NaCl
2.
and PEG
Sour
orange
Cleopatra
mandarin
Treatment
X lower
X lower
(-MPa)
Mean1
than NS
Mean
than NS
NS control
104 ay
0
106 a
0
NaCl (0.10)
64 b
39
79 b
26
NaCl (0.20)
52 c
50
71 c
33
NaCl (0.35)
48 cd
54
55 d
48
PEG (0.10)
60 b
42
63 c
41
PEG (0.20)
41 de
61
54 d
49
PEG (0.35^
38 e
64
43 e
59
Mean of 7 plants.
Mean separation within columns by Duncan's Multiple Range
Test, 0.05 level.
109
Table 32. Total leaf area (cm ) of seedlings of 2
rootstocks grown for 6 months under different
NaCl and PEG concentrations — Experiment 2.
Sour
orange
Treatment
% lower
(-MPa)
Mean*
than NS
NS control
3595 ay
0
NaCl (0.10)
2002 b
44
NaCl (0.20)
1381 c
62
NaCl (0.35)
881 d
76
PEG (0.10)
1019 cd
72
PEG (0.20)
431 e
88
PEG (0.35)
213 e
94
Cleopatra mandarin
% lower
Mean than NS
3601 a
0
1782 b
51
1421 be
61
725 de
80
913 cd
75
498 de
86
294 e
92
Mean of 7 plants.
yMean separation within columns by Duncan's Multiple Range
Test, 0.05 level.
110
Table 33. Specific leaf weight (mg/cm ) of seedlings of 2
rootstocks grown for 6 months under different
NaCl and PEG concentrations — Experiment 2.'
Sour
orange
Cleopatra
Fresh wt
mandarin
Fresh wt
Dry wt
Dry wt
Treatment
basis
basis
basis
basis
NS control
25.1 ay
9.0 b
18.7 b
8.3 c
NaCl (0.10)
27.5 a
9.5 b
18.9 b
8.4 c
NaCl (0.20)
27.9 a
9.6 b
19.1 b
8.5 c
NaCl (0.35)
28.2 a
9.6 b
21.1 a
8.7 c
PEG (0.10)
18.2 b
11.2 a
18.3 b
10.1 b
PEG (0.20)
17.4 be
11.5 a
18.2 b
12.1 a
PEG (0.35)
14.4 c
11.3 a
18.1 b
11.9 a
zMean of 7 plants.
yMean separation within columns by Duncan's Multiple Range
Test, 0.05 level.
Ill
Rootstock
CM SO SC CC RL ML PT
</)
z
c
ta
- 100
0)
o
a? 200
« 300
■a
X
o 40°
o
cc
o
■= 500
a. 600
n
wnn
Fig. 20. Effect of 3 NaCl concentrations (a = -0.10 MPa,
b = -0.20 MPa, c = -0.35 MPa) on the osmotic
potential of root exudate collected from seedlings
of 7 citrus rootstocks.
112
E
o
<x>
<J
c
2
o
D
XD
C
o
O
.50
.40
.30
■a 20
Ta
E
o
oo 10
Sour orange
NaCI
7 11 15
7 11 15
Time (hr)
//
NS
■\ / «0.10
^■-■-■-■0.20
a-I^a-a-a~a0-35
j i i < i
11 15
Fig. 21. Relationship of time of day to stomatal conductance of
sour orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCI during 3
consecutive days. Measurements were started on
April 9, 1986.
113
o
c
o
O
.40
8 -30
.20
CO
T3 -10
E
o
00
Cleopatra mandarin
NaCI
j i i i i i
11 15
#7
a-a-JK^X *£**£] §;1°
I I I I L
11 15
Time (hr)
-/A
I l I I L
11 15
Fig. 22. Relationship of time of day to stomatal conductance
of Cleopatra mandarin seedlings irrigated with
nutrient solution containing no salt (NS) or with
added NaCI during 3 consecutive days. Measurements
were started on April 9, 1986.
114
jr .50
.40
E
o
^^
OJ
O
c
TO
o .30
"a
c
o
o
TS -20
E
o
w .10
Sour orange
PEG
fife**8
2-o.
11 15
-J f—x — i — i — i — i — Ly/-i i i i i i U-oo
J 1 I I I L
11 15
Time (hr)
11 15
Fig. 23. Relationship of time of day to stomatal conductance of
sour orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added PEG during 3
consecutive days. Measurements were started on
April 9, 1986.
115
E
o
^^
<D
O
c
CO
o
"O
c
o
O
« .10
.40
.30
.20
r
o-o-o^g^a
a-a-g^A
-A-
Cleopatra mandarin.
PEG
7 11 15
NS
0.10
0.20
0.35
Time (hr)
Fig. 24. Relationship of time of day to stomatal conductance of
Cleopatra mandarin seedlings irrigated with nutrient
solution containing no salt (NS) or with added PEG
during 3 consecutive days. Measurements were started
on April 9, 1986.
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BIOGRAPHICAL SKETCH
Mongi Zekri was born in Kerkennah, Tunisia, on December 3, 1955. He
received his secondary education and graduated from Chott-Mariem High
School, Sousse, in June 1972. He obtained the diplome of Baccalaureat
of Science in September 1972.
He entered the National Institute of Agronomy in Tunis in October
1972 and graduated with the degree of Bachelor of Engineering in June
1976. Upon graduation, he was employed by the Office of Cereals under
the Ministry of Agriculture as an inspector and a researcher on wheat
for two and a half years. He served in the Army from March 1977 to
March 1978 and obtained the rank of lieutenant in August 1977.
He was awarded a scholarship to pursue graduate studies in the
United States. He attended the Intensive English Program in the spring
and summer of 1980 at the University of Missouri-Columbia. In the fall
of 1980, he enrolled at the University of Florida as a graduate student
in the Fruit Crops Department and earned the degree of Master of Science
in April 1984.
He completed his work toward the degree of Doctor of Philosophy in
December 1987.
He is married to the former Leila Atia. They have one daughter,
Dhoha .
132
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degreeof
Doctor of Philosophy.
Lawrence R. Parsons, Chairman
Associate Professor of Horticultural
Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
William S. Castle
Professor of Horticultural Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Robert C. J. Koo
Professor of Horticultural Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Donald L.~ Myhre
Professor of Soil Sd
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
AIL fi.3v^d
" trla f
Allen G. Smajstrla
Associate Professor of Agricultural
Engineering
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy. ^
December 1987 XwA &■ JTVk/
Dean, Colleg~e of Agriculture
Dean, Graduate
UNIVERSITY OF FLORIDA
ill
3 1262 08553 4526
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